UNIVERSIDADE DE COIMBRA FACULDADE DE MEDICINA Gisela … · 2020. 5. 25. · 30 June, 2012 [Poster...
Transcript of UNIVERSIDADE DE COIMBRA FACULDADE DE MEDICINA Gisela … · 2020. 5. 25. · 30 June, 2012 [Poster...
UNIVERSIDADE DE COIMBRA
FACULDADE DE MEDICINA
Gisela Filipa Assunção Santos
Establishing the validity of glycoursodeoxycholic acid as a coadjuvant of
temozolomide therapy in gliomas
Dissertação apresentada para a obtenção do Grau de Mestre
em Investigação Biomédica, pela Universidade
Coimbra, Faculdade de Medicina
Orientadora:
Profª. Doutora Dora Maria Tuna de Oliveira Brites (FF/UL)
Co-orientadores:
Doutora Ana Sofia Iria Azeredo Falcão de Jesus
(FF/UL)
Prof. Doutor João José Oliveira Malva
(FM/UC)
Coimbra, 2012
The studies presented on this Master Thesis were conducted in the research group
“Neuron Glia Biology in Health & Disease”, from Research Institute for Medicines and
Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon,
under the internal supervision of Dora Brites and co-supervised by Ana Sofia Falcão.
This work was funded by PEst-OE/SAU/UI4013/2011 to iMed.UL, from Fundação
para a Ciência e Tecnologia (FCT), Portugal.
The results here obtained were performed in collaboration with the Master Student
Cátia Gomes, on the thesis entitled “Cues for cancer stem cells origin”, in Molecular
Genetics and Biomedicine, Faculty of Sciences and Technology, New University of
Lisbon (supervised by Dora Brites and co-supervised by Ana Sofia Falcão).
Part of the results discussed in this thesis were presented in the following
publications/ communications:
Torrado E, Gomes C, Santos G, Brites D, Falcão AS. Directing mouse embryonic
neurosphere differentiation towards nerve cell lineages. Experimental Neurology
2012 [submitted];
Gomes C, Santos G, Torrado E, Falcão AS, Brites D. Applying Neural Stem Cell
Biology To Brain Tumor Research: New Cues For Gliomagenesis In The Elderly. 26ª
Reunião do Grupo de Estudos do Envelhecimento Cerebral e Demência, Tomar, 29-
30 June, 2012 [Poster comunication];
Santos G, Gomes C, Torrado E, Falcão AS, Lopes MC, Brites D. Multidrug
Resistance-Associated Protein 1 Inhibition As A Way To Enhance Cytotoxicity Of
Temozolomide In Mouse Glioma Cells. 26ª Reunião do Grupo de Estudos do
Envelhecimento Cerebral e Demência, Tomar, June 29-30, 2012 [Poster
comunication].
Agradecimentos
As minhas primeiras palavras de agradecimento vão naturalmente para a
Professsora Dora Brites, orientadora deste trabalho. Agradeço-lhe por me ter
recebido no grupo e me ter dado a conhecer novos caminhos no mundo da ciência,
assim como todo o conhecimento que me transmitiu. Agradeço-lhe ainda o
encorajamento e o apoio ao longo deste último ano, sendo que os seus elevados
padrões de rigor científico e exigência, assim como capacidade de raciocíonio
científico e o seu espirito crítico contribuíram de uma forma muito positiva na
orientação deste trabalho. Agradeço também a disponibilidade e espero ter
correspondido às suas expectativas!
A ti, Sofia, agradeço não só a disponiblidade e compreensão, mas também a
constante ajuda e motivação. Os teus conhecimentos e apoio foram fundamentais
para a progressão deste trabalho, principalmente quando as coisas corriam menos
bem, permitindo ultrapassar as dificuldades que foram surgindo. Um muito obrigado
ainda pelo carinho e preocupação demonstrada, quer a nível profissional, quer a
nível pessoal. A tua simpatia e boa disposição contagiantes tornaram o facto de ter
trabalhado contigo e ter estado sob tua co-orientação numa experiência muito
gratificante!
Agradeço ainda ao Professor João Malva, co-orienteador interno.
Independentemente de não ter tido uma co-orientação activa, sempre se mostrou
disponível e manisfestou grande interesse pelo trabalho realizado.
Não podia deixar de agradecer à Professora Celeste por me ter recebido no seu
grupo para realizar parte deste trabalho. Agradeço-lhe igualmente, simpatia e a
preocupação na tentativa que tudo corresse da melhor forma durante a minha
estadia em Coimbra.
Agradeço também ao Professor Rui Silva e à Professora Alexandra Brito por todos
os conhecimentos e conselhos científicos que foram transmitindo ao longo deste
ano. Adelaide, o teu imenso conhecimento (não só científico) e dedicação são
completamente inspiradores. Agradeço-te pelo tempo que dispendeste sempre que
precisei, assim como pelos pontos de vista e todas as sugestões que me ajudaram
quando a Sofia não estava presente, tendo também contribuindo para a progressão
deste trabalho. Agradeço ainda a forma como me receberam no grupo.
Agradecimentos
6
Às restantes meninas do grupo Neuron Glia Biology in Health & Disease e às
pessoas que tornaram a passagem por Coimbra uma experiência única, um muito
obrigada pela amizade e por todos os momentos partilhados (tanto os bons, como
os menos bons). Foi uma honra para mim poder conhecer-vos.
Aos meus amigos, um obrigada muito especial. Poderia enumerar todas as razões,
mas cada um de vocês sabe a importância que tem na minha vida (espero eu!).
Apesar de nem todos os momentos terem sido os mais fáceis, agradeço aos meus
pais e ao meu irmão pelo papel que tiveram na minha vida e pelas decisões que me
fizeram tomar, que me trouxeram até aqui.
Mara, a ti, fica o agradecimento mais especial. Muito obrigada por me fazeres sorrir
independentemente da quantidade e gravidade dos problemas que tenho.
Contents
Abbreviations
Abstract
Resumo
Chapter I - Introduction
1. Brain tumors
1.1. Classification of gliomas
1.2. Epidemiology of gliomas
1.3. Signaling pathways regulating gliomagenesis
1.4. Tumorigenic properties
1.4.1. Tumor cell invasion
1.4.2. Angiogenesis
1.4.3. Resistance to chemoradiotherapies
1.4.4. Autophagy
1.5. Diagnosis and treatment
1.5.1. Temozolomide as a chemotherapeutic agent
1.5.2. Ursodeoxycholic acid (UDCA) and its glyco- (GUDCA) and tauro-
(TUDCA) conjugated species
2. Neural stem cells (NSCs)
2.1. NSC in the developing and adult brain
2.2. Applying NSC biology to glioma research: the brain tumor stem cells
(BTSCs) hypothesis
2.2.1. The origin of BTSCs
2.2.2. Therapeutic perspectives
Chapter II – Objectives
Chapter III – Materials and methods
1. Cell cultures
1.1. GL261 mouse glioma cell line
1.2. Primary neurosphere culture of mouse brain cortex at E15 and
induction of astrocyte differentiation
2. Characterization of the mouse glioma cell line GL261
2.1. Characterization of the GL261 cells by immunocytochemistry
2.2. Characterization of the GL261 cells by flow cytometry
3. Characterization of tumor-associated factors
3.1. MMPs activity
xiii
xiv
xvii
1
3
3
5
5
7
8
9
11
12
13
14
16
17
17
18
20
22
25
29
31
31
31
32
32
32
33
33
Contents
8
3.2. S100B assay
3.3. Expression of tumor-associated factors
4. Cell treatments
4.1. Cell viability
4.2. Cell cycle progression
4.3. Expression of CXCR4
Chapter IV – Results and discussion
1. Characterization of the mouse glioma cell line GL261
2. Characterization of common features between GL261 glioma cells and
differentiating astrocytes from neural stem cells
2.1. Invasion ability
2.2. Angiogenesis
2.3. Multidrug resistance
2.4. Autophagy
3. Effects of a combined anticancer strategy on GL261 cell viability and
cell cycle
3.1. Effect of TMZ on glioma cells viability
3.2. Effect of TMZ, GUDCA and TMZ+GUDCA on glioma cells viability
and cell cycle
3.3. Effect of TMZ, MK-571 and TMZ+MK571 on glioma cells viability
and cell cycle
4. Effect of GUDCA and MK-571 in tumor cell migration
Chapter V – Concluding remarks
Chapter VI - References
33
34
34
35
35
35
37
39
43
44
46
48
49
51
51
52
55
57
63
67
Figures
Fig. I.1. – Histophatologic progression of infiltrating astrocytoma to
glioblastoma multiforme (GBM) according to the WHO classification
Fig. I.2. - Pathways that mediate the development of glioblastoma
Fig. I.3. - Multidrug resistance mechanism of the multidrug resistance-
associated protein 1 (Mrp1)
Fig. I.4. - Chemical structure of temozolomide (TMZ) and of its metabolites
Fig. I.5. – Mechanisms of activity of temozolomide (TMZ) as an enhancer
therapeutics
Fig. I.6. – Characteristics of brain tumor stem cells (BTSCs)
Fig. I.7. – Possible origins of brain tumor stem cells
Fig. IV.1. Characterization of the glioma cell line GL261
Fig. IV.2. Metalloproteinase (MMP)-2 and MMP-9 activities in GL261 glioma
cells at 3, 5 and 7 days in vitro and in differentiating astrocytes from
neurospheres (NS) during 3 and 7 DIV
Fig. IV.3. S100B release from GL261 glioma cells at 3, 5 and 7 days in vitro
and in differentiating astrocytes from neurospheres (NS) during 3 and 7 DIV
Fig. IV.4. VEGF expression in GL261 glioma cells at 3, 5 and 7 days in vitro
and in differentiating astrocytes from neurospheres (NS) during 3 and 7 DIV
Fig. IV.5. Mrp1 expression in GL261 glioma cells at 3, 5 and 7 days in vitro
and in differentiating astrocytes from neurospheres (NS) during 3 and 7 DIV.)
Fig. IV.6. LC3II/I expression in glioma cells at 5 days in vitro and in
differentiating astrocytes from neurospheres (NS) during 3 and 7 DIV.
Fig. IV.7. Effect of temozolomide (TMZ) addition in glioma cells viability.
Fig. IV.8. Cell viability of GL261 cells in the absence (control) or in the
presence of temozolomide (TMZ), glycoursodeoxycholic acid (GUDCA), and
TMZ+GUDCA
Fig. IV.9 Cell viability of GL261 cells in the absence (control) or in the
presence of temozolomide (TMZ), Mrp1 inhibitor (MK-571) and TMZ+MK-571
Fig. IV.10. CXCR4 expression in glioma cells at 3, 5 and 7 days in vitro in the
absence (control) or in the presence of either glycoursodeoxycholic acid
(GUDCA) ot the Mrp1 inhibitor MK-571
Fig. V.1. Summary of GL261 cell line characterization
4
7
11
14
15
19
21
43
45
46
47
48
50
52
53
56
58
64
Tables
12
Table IV.1. Cell cycle analysis of GL261 cells incubated in the presence of
temozolomide (TMZ), glycoursodeoxycholic acid (GUDCA) and
Table IV.2. Cell cycle analysis of GL261 cells incubated in the presence of
temozolomide (TMZ), MK-571 and TMZ+GUDCA, during 24 and 72
Table. V.1. Characterization of common features between GL261 glioma
cells and neurospheres induced to differentiate into astrocytes during 3 and 7
days in vitro
55
57
65
Abbreviations
ABC-transporter
AIC
BTSC
CNS
DIV
DNA
ECM
DMEM
FBS
GBM
GFAP
GLAST
GSH
GUDCA
HIF-1
IDH
LC3
MAP2
MGMT
MMP
MRP1
MTIC
NEP
ATP-binding cassette transporter
Aminoimidazole-4-carboxamide
Brain tumor stem cell
Central nervous system
Days in vitro
Deoxyribonucleic acid
Extracellular matrix
Dulbecco’s modified eagle’s medium
Fetal bovine serum
Glioblastoma multiforme
Glial fibrillary acidic protein
Glutamate aspartate transporter
Glutathione
Glycoursodeoxicholic acid
Hypoxia inducible factor 1
Isocitrate dehydrogenase
Light chain 3
Microtuble-associated protein 2
Methylguanine methyltransferase
Metalloproteinase
Multidrug resistance-associated protein 1
5-(3-methyltriazen-1-yl)imidazole-4-carboxamid
Neuroepithelial progenitor
Abbreviations
xiv
NS
NSC
PBS
PTEN
Rb
RG
RNA
ROS
RT
SDS-PAGE
SDF-1
SGZ
Sox2
SVZ
TBS
TMZ
VEGF
VZ
WHO
Neurosphere
Neural stem cell
Phosphate-buffer saline
Phosphatase and tensin homolog
Retinoblastoma
Radial glia
Ribonucleic acid
Reactive oxygen species
Radiation therapy
Sodium dodecyl sulfate-polyacrilamide gel
Stroma-cell-derived factor 1
Subgranular zone
Sex determining region Y-box2
Subventricular zone
Tris-buffered saline
Temozolomide
Vascular endothelial growth factor
Ventricular zone
World health organization
Abstract
xv
Brain tumors are the second most common neoplasms in children and their
incidence is also relatively high in the adult population, with gliomas accounting for
the majority of cases. So far, the treatment protocols available for gliomas did not
improve the prognosis, mainly due to a phenomenon known as multidrug resistance.
Thus, research has now been aimed to identify the mechanisms leading to
gliomagenesis and it was recently suggested that neural stem/progenitor cell or
early-differentiated cell type lineages might be in the origin of glioma. Therefore, the
main goals of the present work are: a) to identify which developmental stage, in the
neural stem cell (NSC) differentiation process towards astrocytes, is most similar to
the glioma phenotype and b) to find successful adjuvant molecules for temozolomide
(TMZ), a chemotherapeutic agent.
The GL261 mouse glioma cell line was cultured until 7 days in vitro and some
tumor-related factors were determined in glioma cells and compared with astrocytes
differentiated from mouse neural stem/precursor cells (neurospheres, NS). Moreover,
we also evaluated the potential beneficial effect of TMZ treatment in the presence of
the bile acid glycoursodeoxycholic acid (GUDCA) or the multidrug resistance-
associated protein 1 (Mrp1) inhibitor MK-571. Finally, we have assessed the effect of
both GUDCA and MK-571 on the expression of CXCR4, a chemokine receptor.
The analysis of tumor-related factors showed that during GL261 maturation, there
is a decrease on the expression of the vascular endothelial growth factor (VEGF) as
well as on the activity of the matrix metalloproteinases MMP-9 and MMP-2, which is
associated with an increase on S100B release. Also, the Mrp1 presents a peak of
expression at 5 DIV. Although not as evident as we were expecting, the NS
proliferating stage seems to be the phenotype most similar to glioma cells,
suggesting that the origin of glioma might be somehow associated with NSC
malignant transformation. Moreover, TMZ therapy appears to be improved by the
synergistic effect of GUDCA or Mrp1 inhibition, since it was observed a further
reduction on cell viability and cell cycle arrest at the G2/M phase. Conversely,
GUDCA or MK-571 seem to improve the migratory ability of GL261, by the induction
of increase of CXCR4 levels.
Efforts to enlarge knowledge about the pathways implicated on a malignant
alteration during neural development as well as to further understand how to improve
the current therapy, would allow a more specific targeting and consequently, an
increased survival of glioma patients.
Keywords: GL261 glioma cells; glycoursodeoxycholic acid; MK-571; neural stem
cells proliferation and differentiation; temozolomide; tumor-related factors
xvi
Resumo
xvii
Os tumores cerebrais são as segundas neoplasias mais comuns em crianças e a
sua incidência é também relativamente elevada na população adulta, sendo os
gliomas os mais frequentes. Até agora, os tratamentos disponíveis para gliomas não
melhoraram o prognóstico, principalmente devido a um fenómeno conhecido como
resistência a múltiplas drogas. Assim, atualmente a investigação tem como objetivo
identificar os mecanismos que levam à gliomagénese e foi recentemente sugerido
que células estaminais/progenitoras neurais ou no início da diferenciação podem
estar na origem dos gliomas. Desta forma, os principais objetivos do presente
trabalho são: a) identificar qual o estadio de desenvolvimento, no processo de
diferenciação de células estaminais neurais para astrócitos, é mais parecido com o
fenótipo de glioma e b) encontrar moléculas adjuvantes eficazes na terapia com
temozolomida (TMZ), um agente quimioterapêutico.
As células de glioma de ratinho GL261 foram mantidas em cultura durante 7 dias
in vitro e alguns fatores relacionados tumores foram determinados em células de
glioma e comparadas com os astrócitos diferenciados a partir de células
estaminais/progenitoras de ratinho (neuroesferas, NS). Além disso, foram
igualmente avaliados os potenciais efeitos benéficos do tratamento com TMZ na
presença do ácido glicoursodesoxicólico (AGUDC) ou de um inibidor da proteína
associada à resistência a multidrogas (Mrp1), o MK-571. Finalmente, foi avaliado o
efeito quer do GUDCA, quer do MK-571 na expressão de CXCR4, um recetor de
quimocinas.
A análise dos factores relacionados com tumores mostrou que, durante a
maturação das células GL261, há uma diminuição na expressão do factor de
crescimento endotelial vascular (VEGF), bem como na actividade das
metaloproteinases MMP-9 e MMP-2, associado a um aumento da libertação de
S100B. Além disso, a Mrp1 apresenta um pico de expressão ao 5 DIV. Apesar de
não ser tão evidente como esperávamos, o estadio de NS parece ser o fenótipo
mais semelhante com as células de glioma, o que sugere que a origem dos gliomas
pode estar de alguma forma associada à transformação maligna das CEN. Além
disso, a terapia com TMZ parece ser mais eficaz pelo efeito sinergético do GUDCA
ou inibição da Mrp1, uma vez que se observou uma redução na viabilidade celular e
paragem do ciclo celular na fase G2/M. Por outro lado, o GUDCA ou MK-571
parecem melhorar a capacidade de migração das GL261, através da indução do
aumento dos níveis de CXCR4.
Um melhor conhecimento sobre as vias implicadas em alterações malignas
durante o desenvolvimento neural, bem como sobre novas formas de melhorar a
actual terapia, permitirá o uso de esquemas terapêuticos mais direccionados e
Resumo
xviii
específicos e, consequentemente, um aumento da sobrevivência dos pacientes que
apresentam gliomas.
Palavras chave: células de glioma GL261; ácido glicoursodesoxicólico; MK-571;
proliferação e diferenciação de células estaminais neurais; temozolomida; factores
relacionados com tumores
1
CHAPTER I - INTRODUCTION
1
Chapter I - Introduction
3
1. Brain tumors
The term “brain tumor” refers to a group of neoplasms, with high incidence both
in children as well as in the adult population, and on these, they mainly occur in the
elderly (Sutter et al., 2007). Each neoplasm has its own biology, diagnosis and
treatment. However, the clinical presentation, diagnosis and initial treatment are
similar for most tumors.
Nowadays, tumors of the central nervous system (CNS) are mostly classified by
the World Health Organization (WHO) guidelines, which facilitate the communication
throughout the world. A brain tumor can be primary, if the tumor starts in the brain
and secondary (or metastatic), if it results from somewhere else in the body (ABTA,
2010).
1.1. Classification of gliomas
Gliomas, the most common form of primary brain tumors are characterized by a
great heterogeneity both histologically and clinically, as well as by diverse grades of
malignancy. The most recent classification of gliomas was described by WHO in
2007 based mainly on three parameters: cell type, malignancy grade and tumor
location (Louis et al., 2007).
a) Classification Based on Cell Type
This type of classification is based on the histological characteristics of the cells,
according to the phenotypic and morphologic similarities of the tumor cells with those
of different types of glial cells, such as astrocytes, oligodendrocytes and ependymal
cells. Thus, gliomas can be classified as astrocytomas, oligodendrogliomas,
ependymomas and also oligoastrocytomas (or mixed gliomas). Among all these
types of tumors, astrocytomas are the most common.
b) Classification Based on Malignancy Grade
Grading of tumors facilitate the treatment and the prediction of their outcome. The
grade indicates its degree of malignancy and it is assigned based on the tumor’s
microscopic examination using criteria: like similarity to normal cells (atypia), rate of
growth (mitotic index), indication of uncontrolled growth, dead tumor cells in the
center of the tumor (necrosis), potential for invasion and/or spread (infiltration) based
on whether or not it has a definitive margin (diffuse or focal), and blood supply
(vascularization) (Fig.I.1). When gliomas contain several grades of cells, the grade is
Chapter I - Introduction
4
determined by the highest or most malignant grade of cells, even if most of the tumor
is a lower grade kind.
Fig. I.2. – Histophatologic progression of infiltrating astrocytoma to glioblastoma
multiforme (GBM) according to the WHO classification. The normal brain white matter
(A), presents blood vessels (arrows) and cell density similar with an infiltrating astrocytoma
grade II (B). Arrowhead shows that tumor cells of this grade II tumor can be found near the
CNS parenchyma. Anaplastic astrocytoma (AA; grade III; C) is characterized by a higher
number of cell and blood vessels density, which are often dilated or with thickness walls
(arrows). AA cells also present an atypical morphology and some mitotic cells can be found.
GBM (D) shows necrotic zones with pseudopalisading tumor cells (asterisk within necrotic
center), which are usually surrounded by microvascular hyperplasia and vascular glomerules
proliferation (arrow). From Brat et al. (2003).
Grade I gliomas are benign and are typically related with long-term survival. The
tumors exhibit a slow grow and a limited cell proliferation potential and have an
almost normal appearance when viewed through a microscope. Surgery alone might
be an effective treatment for these tumors. Grade II tumors have a relatively slow
growing rate, a low-level of proliferative activity and a slightly abnormal microscopic
appearance. They are in general infiltrative and some can recur as a higher-grade
glioma. Grade III tumors are, by definition, malignant. The cells of a grade III tumor
are actively reproducing abnormal cells, which grow into nearby normal brain tissue.
These tumors tend to recur, often as a higher grade. In most cases, it is necessary to
receive adjuvant radiation and/or chemotherapy. The tumors of grade IV are the
most malignant. In addition to the strange appearance, they have a high mitotic
activity and can easily spread into the surrounding normal brain tissue. These tumors
have also an enormous ability to form new blood vessels so they can maintain their
rapid growth. Necrosis zones are usually associated with a rapid evolution and fatal
outcome. The glioblastoma multiforme (GBM), sometimes referred only as
A B C D
Chapter I - Introduction
5
glioblastoma, is the most common between grade IV tumor (Louis et al., 2007;
Ohgaki, 2009).
c) Classification Based on Tumor Location
Gliomas can also be classified regarding their location, whether above or below
the tentorium, a membrane that separates the cerebrum (above) from the cerebellum
(bellow). Hence, they are defined as supratentorial, which develop above the
tentorium, and as infratentorial, which develop below the tentorium. The
supratentorial and the infratentorial gliomas correspond to 70% of the tumors in
adults and children (Louis et al., 2007).
1.2. Epidemiology of gliomas
The peak of gliomas onset is around 50-55 years, which makes them a strongly
age-related pathology. The incidence of brain tumors tends to be highest in
developed and industrialized countries. In Western Europe, North America and
Australia there are about to 6-11 new cases of primary intracranial tumors per 100
man individuals and to 4-11 new cases in the women population (Ohgaki and
Kleihues, 2005; Ohgaki, 2009). Ethnic differences in the vulnerability to develop of
brain tumors cannot be excluded. Caucasians are more susceptible than African or
Asian people. Some reports indicate that the incidence rate of gliomas is
approximately twice in whites when compared to blacks. In addition, in Japan,
gliomas are about half as frequent as in the United Sates of America (Ohgaki and
Kleihues, 2005; Ohgaki, 2009).
With the exception of pilocytic astrocytoma (WHO grade I), survival of glioma
patients is still poor and one of the factors for this is the older age at diagnosis.
Mortality rates from CNS tumors are similar to the incidence rate, i.e., around to 4-7
cases per 100,000 persons per year in men and to 3-5 cases in women, throughout
the geographical areas referred above (Ohgaki and Kleihues, 2005; Ohgaki, 2009).
1.3. Signaling pathways regulating gliomagenesis
It is recognized that morphological changes during the malignant transformation,
reflect the sequential acquisition of genetic alterations. Although primary and
secondary tumors differ on the genetic level in many ways, there are some common
genetic abnormalities, which are considered as hallmarks of gliomas. So far, a
variety of studies have identified DNA copy number alterations and mutations as
Chapter I - Introduction
6
recurrent events on gliomagenesis, suggesting the involvement of tumor suppressor
genes and oncogenes (Fig. I.2) in tumor initiation and progression (Furnari et al.,
2007; Ohgaki, 2009).
The first studies identified the existence of mutations in the epidermal growth
factor receptor (EGFR) gene, encoding the receptor for the epidermal growth factor
(EGF) (Van Meir et al., 2010). Mutation in EGFR glioma enhances tumorigenic
behavior by reducing apoptosis and increasing proliferation (Furnari et al., 2007).
Later, further analysis identified the p53 tumor suppressor gene, as important for,
which is involved in the regulation of cell cycle progression and apoptosis in
response to a wide variety of stress signals, including DNA damage. Upon exposure
to genotoxic agents, p53 is stabilized, accumulates in the nucleus, binds and
transcriptionally regulates the promoters of potential effector genes. Since p53
function is critical for normal cell growth and development, its activity is tightly
regulated by phosphorylation, which is the first step to induce stabilization of p53
(Carmo et al., 2011). This pathway is nearly invariably altered in sporadic gliomas
and frequent in the beginning of secondary glioblastomas (Furnari et al., 2007;
Carmo et al., 2011). The next genes discovered were the p16 cell cycle inhibitor, and
the phosphatase and tensin homolog (PTEN), acting both as tumor suppressors. The
p16 is responsible for the slow down of the cell cycle progression, whereas PTEN is
a negative regulator of the phosphoinositide 3–kinase (PI3K) pathway, a major
signaling pathway that stimulates cellular proliferation in response to growth factor
stimulation (Westphal and Lamszus, 2011). Inactivation of PTEN is associated with
increased angiogenesis, a parallel process in the progression of high grade gliomas
(Furnari et al., 2007). In fact, PTEN mediates a variety of biological functions like
apoptosis, inflammation and immunity (Westphal and Lamszus, 2011).
Recently, some mutations were identified at the level of the genes encoding
isocitrate dehydrogenase 1 (IDH1) (and to a lesser extent IDH2) and retinoblastoma
(RB) in lower grade gliomas and in a subset of glioblastomas. IDH1 mutation is
associated with longer survival of patients with secondary glioblastoma and one
consequence of its raised expression is an altered pattern of DNA methylation in
gene promoter regions, leading to epigenetic silencing (Westphal and Lamszus,
2011). RB blocks proliferation by binding and sequestering the E2F family of
transcription factors, which prevents the activation of essential genes for progression
through the cell cycle (Furnari et al., 2007). Moreover, mutations in the ERBB2 gene
have also been found as recurrent event in primary glioblastomas (Westphal and
Lamszus, 2011).
Chapter I - Introduction
7
Fig. I.2. - Pathways that mediate the development of glioblastoma. Distinct molecular
alterations correlate with the clinical development of gliomas. EGFR, endothelial growth factor
receptor; IDH1, Isocitrate dehydrogenase 1; LOH, Loss of heterozygosity 10q; P16 INK4a
,
Cyclin-dependent kinase inhibitor 2A; PTEN, Phosphatase and tensin homolog; TP53, tumor
protein 53. Adapted from Sulman (2009).
Although histologically indistinguishable, GBM can occur in different age groups
and present distinct genetics alterations affecting similar pathways. The
understanding and identification of these alterations will assist a more correct
diagnosis.
1.4. Tumorigenic properties
The tumorigenic properties that are the most responsible for the initiation and
maintenance of tumor include tumor cell invasion, angiogenesis, resistance to
therapy and autophagy, which will be further discussed.
Glioma cell of origin
Low-grade astrocytoma
TP53 mutation (~ 60%)
Anaplastic astrocytoma
TP53 mutation (~ 50%)
Secondary glioblastoma
LOH 10q (~ 60%)
EGFR amplification (~ 5%)
P16INK4a
deletion (~ 20%)
TP53 mutation (~ 70%)
PTEN mutation (~ 5%)
IDH1 (? %)
Primary glioblastoma
LOH 10q (~ 70%)
EGFR amplification (~ 30%)
P16INK4a
deletion (~ 30%)
TP53 mutation (~ 30%)
PTEN mutation (~ 30%)
IDH1 (? %)
Chapter I - Introduction
8
1.4.1. Tumor cell invasion
The infiltrative nature of tumors makes curative surgical resection nearly
impossible and contributes to the poor prognosis and short median survival of
patients (Choe et al., 2002).
Invasion of tumor cells into adjacent brain structures occurs through the
activation of matrix metalloproteinases (MMPs). MMPs are a family of zinc-
dependent endopeptidases that mediate the degradation of protein components of
the extracellular matrix (ECM) and of basement membranes (Choe et al., 2002;
Hagemann et al., 2012). Degradation of the ECM by MMPs not only enhances tumor
invasion, but also affects tumor cell behavior and leads to cancer progression. MMPs
can be classified as collagenases (MMP1, MMP8 and MMP13), stromelysins (MMP3,
MMP10, MMP11, MMP7 and MMP26), gelatinases (MMP2 and MMP9) and as
membrane-type (MMP14, MMP15, MMP16, MMP17, MMP24 and MMP25) (Rao,
2003).
MMPs enhance tumor-cell invasion and migration by degrading ECM proteins,
activating signal transduction cascades that promote motility and solubilizing
extracellular matrix-bound growth factors, in particular by cleaving laminin-5 (Choe et
al., 2002; Rao, 2003; Hagemann et al., 2012). In fact, it was observed that
interference with MMP-9 and one of its upstream regulators by RNA interference
lead to a reduction in tumor growth and invasion in a mouse model. MMP-9, MMP-2
and its activator MMP-14 are involved in migration and invasion of human GBM cells
and the first clinical trials using the MMP inhibitor, marimastat, in combination with
chemotherapy have recently been performed in GBM patients (Hagemann et al.,
2012).
In addition, MMPs also play a central role in a number of physiological processes,
such as cell growth and development (by cleaving and activating some growth
factors, as the transforming growth factor-β), differentiation, angiogenesis (by
increasing the bioavailability of pro-angiogenic growth factors) and apoptosis (Lu et
al., 2010; Ponnala et al., 2011). Very recently, it was published a review that
summarizes the currently available data on the expression of MMPs in human
glioblastomas (Hagemann et al., 2012).
Besides MMPs, S100B is also related with tumor cell invasion. This protein is a
member of a multigenic family of Ca2+-binding protein of the EF-hand type, and is
located diffusely in the cytoplasm and associated with membranes and certain
cytoskeleton elements (Brozzi et al., 2009; Zhang et al., 2011). S100B has been
implicated in the regulation of both intracellular and extracellular activities, such as
Chapter I - Introduction
9
regulation of the state of microtubules assembly and type III intermediate filaments,
some enzyme activities, and cell proliferation. High levels of S100B are found in
certain cancer cells, reason why it has been proposed that it contributes to
tumorigenesis by inhibiting the function of p53 protein and by regulating cell
proliferation and differentiation by stimulation of kinases activation (Brozzi et al.,
2009; Zhang et al., 2011). Being a chemotactic molecule, S100B protein stimulates
microglia migration via RAGE-dependent up-regulation of chemokine expression and
release (Bianchi et al., 2011). Thus, we can hypothesize that this molecule may
perform a pivotal function in tumor cell invasion and metastasis.
Other molecules also associated to tumor cells invasion are chemokines, which
are a family of chemotatic cytokines involved in multiple biological functions, as
leukocyte migration, hematopoiesis, mitosis, apoptosis, survival, angiogenesis and
tumor cell growth (Carmo et al., 2010; Calatozzolo et al., 2011). The most important
chemokine associated with tumorigenesis process and metastasis is the stroma-cell-
derived factor 1 (SDF-1/CXCL12) and its receptor, the CXCR4, a G-protein, which
expression is upregulated by hypoxia, via hypoxia-inducible factor 1 (HIF-1α) and
vascular endothelial growth factor (VEGF) (Calatozzolo et al., 2011). The activation
of CXCR4 by CXCL12 regulates numerous essential processes such as cardiac and
neuronal development, stem cell motility, and as a pro-angiogenic factor (Calatozzolo
et al., 2011). Regarding brain tumors, it has been shown that both CXCR4 and
CXCL12 were overexpressed when compared with normal tissue, predominantly in
necrosis areas and angiogenesis (Calatozzolo et al., 2011), what is correlated with
the infiltrative extension of the tumor (Carmo et al., 2010). In vivo and in vitro studies
demonstrated that CXCL12 promotes tumor growth and inhibits apoptosis through
Erk1/2 and Akt pathways and also mediated glioma chemotaxis (Calatozzolo et al.,
2011). On the other hand, CXCR4 expression in malignant gliomas has been
associated with poor prognosis in patients and mouse models (Calatozzolo et al.,
2011). Thus, the importance that this CXCL12/CXCR4 axis has in tumorigenesis
makes it a great therapeutic target in glioma treatment.
1.4.2. Angiogenesis
Studies support that angiogenesis, the formation of new vessels from pre-existing
ones, is required for tumor growth. Thus, during the last three decades, intensive
research has been performed to characterize the angiogenesis process and many
angiogenesis-related factors or genes have been identified (Jouanneau, 2008).
Chapter I - Introduction
10
Angiogenesis is characterized by a series of steps including degradation of the
basement membrane, endothelial cell proliferation, invasion of the surrounding
stroma and structural reorganization into a novel functional vascular network through
the recruitment of perivascular supporting cells (Jansen et al., 2004). The complexity
of the process implies the involvement of multiple regulatory factors, such as growth
factors, adhesion molecules and matrix-degrading enzymes. Malignant gliomas
exhibit many vessel-related pathological features (Yamanaka and Saya, 2009).
These features include marked endothelial proliferation, and tortuous disorganized
vessels of higher permeability, larger diameters and thicker basement membranes
than vessels found in normal tissues. Aberrant microvasculature typically appears as
glomeruloid tufts, proliferations of microvessels consisting of multilayered mitotically
active endothelial and perivascular cells (Jain et al., 2007).
The most important growth factor in angiogenesis is VEGF, which has highly
specific mitogenic and chemotactic activity on endothelial cells (Kargiotis et al.,
2006). Up-regulation of VEGF seems to be triggered by hypoxia through HIF-1 and
mediated by two mechanisms (Jansen et al., 2004; Kargiotis et al., 2006). Through
multiple regulatory mechanisms, HIF acts as a delicate sensor leading to a rapidly
cell response to changes in environmental levels of oxygenation (Kaur et al., 2005).
First, hypoxia induces the activation of VEGF gene transcription through an HIF-
dependent mechanism, mediated by HIF-1 binding to the VEGF promoter, resulting
in increased gene transcription. The second mechanism upregulates VEGF mRNA
levels by regulating mRNA stability. Regulation is finely tuned to the availability of
oxygen because HIF-1a, the oxygen-sensitive subunit of the HIF-1 complex, is stable
in hypoxia (Jansen et al., 2004). VEGF is predominantly located in the
pseudopalisading cells surrounding hypoxic/necrotic foci, which is likely due to
hypoxic induction. In fact, besides abundant microvessels, regional necrosis is
another common pathological feature in glioma tissues and emerging evidence has
suggested that hypoxia is an important modulator in the process of glioma
angiogenesis (Jensen, 2009). Thus, due to obvious and aggressive vascular
proliferation and very poor prognosis, many antiangiogenic drugs were rushed for
approval in clinical trials for glioma patients. Unfortunately, under experimental and
clinical conditions, antiangiogenic therapy has led to increased invasion and higher
recurrence rates (Thurston and Kitajewski, 2008).
Chapter I - Introduction
11
1.4.3. Resistance to chemoradiotherapies
Although chemo and radiotherapy remain the adjuvant treatments of brain
cancer, these treatments fail to cure the majority of patients mainly due to
chemoresistance. Several mechanisms may contribute to the development of
therapeutic resistance, including cell intrinsic factors, selection of resistant genetic
subclones, and microenvironmental factors.
ATP-binding cassette (ABC)-transporters are transmembrane proteins that utilize
ATP hydrolysis to transport substrates from the intracellular to the extracellular
compartment (Atkinson et al., 2009), acting as drug efflux pumps and decreasing the
intracellular levels of various cytotoxic agents (Benyahia et al., 2004; Lebedeva et al.,
2011). The multidrug resistance-associated protein 1 (MRP1/ABCC1) is a member of
a subfamily of the ABC-transporters superfamily. It was discovered by Cole et al.
(Cole et al., 1992), that described it as responsible for multidrug resistance of tumors
(Begley, 2004). In addition to its ability to confer resistance in tumor cells, multi-
resistance protein 1 (MRP1) is ubiquitously expressed in normal tissues and is a
primary active transporter of glutathione (GSH), although it also transports
unmodified xenobiotics that often require GSH (Fig. I.3) (Leslie et al., 2001). In
untreated gliomas, an overexpression of MRP1 has been reported in about 70% of
cases, with a higher expression in high-grade gliomas, particularly glioblastoma
(Benyahia et al., 2004; Lebedeva et al., 2011).
Fig. I.3. - Multidrug resistance mechanism of the multidrug resistance-associated
protein 1 (Mrp1). Mrp1, a transmembrane protein, functions as an ATP-dependent efflux
pump by carrying cytotoxic drugs out from brain cells mediated by the conjugation with
glutathione (GSH), particularly in glioma cells. Adapted from Bredel et al. (2001).
Chapter I - Introduction
12
Besides MRP1 phenotype, resistance to chemotherapy is often caused by
elevated levels of enzymes involved in intracellular drug mechanism, including
MGMT, as already described, contributing to resistance to alkylating agents.
1.4.4. Autophagy
Autophagy, also known as the programmed cell death type II, is a conserved
process that degrades and recycles organelles and portions within cytosol. The
intracellular molecules and organelles, such as endoplasmic reticulum, mitochondria,
and peroxisomes, are sequestered into double-membrane structures called
autophagosomes (autophagic vesicles) (Fan et al., 2010). The C-terminal fragment
of microtubule-associated protein light chain 3 (LC3, which is essential for
autophagy) is cleaved to a cytosolic form LC3-I, which is further converted to LC3-II,
a 16-kDa protein that localizes into autophagosomal membranes. Autophagosomes
then fuse with lysosomes, forming autophagolysosomes, which promote the
degradation of intracellular contents by lysosomal enzymes (Fan et al., 2010; Lin et
al., 2012). Autophagy thus enables the cell to eliminate and recycle proteins or
organelles to sustain metabolism and can be recognized in part by formation of LC3-
II punctae (Fan et al., 2010), since the amount of LC3-II correlates with the number
of autophagosomes. Therefore, LC3-II is considered an autophagy marker (Lin et al.,
2012).
This type of cell death is also highly adaptable and can be modified to digest
specific cargoes to bring about selective effects in response to numerous forms of
intracellular and extracellular stress. It is not a surprise, therefore, that autophagy
has a fundamental role in cancer and that perturbations in autophagy can contribute
to malignant disease. However, there are conflicting reports suggesting that
autophagy can be both oncogenic and tumor suppressive, perhaps indicating that
autophagy has different roles at different stages of tumor development. Recent data
point out that this process may play a critical role in the benign to malignant transition
that is also central to the initiation of metastasis (Macintosh et al., 2012).
1.5. Diagnosis and treatment
The classification of a tumor stage determines if it has spread beyond the site of
its origin and this information often influences treatment recommendations and
prognosis. The first steps of the diagnosis consist in making the medical history and
a basic neurological exam, which analyzes diverse parameters, including memory.
Chapter I - Introduction
13
Thereafter, if the result is suspicious, additional testing as scans, like Magnetic
Resonance Imaging (MRI) and Computerized Tomography (CT) or Positron
Emission Tomography (PET), x-rays or laboratory tests are performed. During and
after treatment, it is recommended to repeat these tests in order to follow the
evolution or stage of the disease (Omay and Vogelbaum, 2009).
Until recently, treatment decisions regarding malignant gliomas began only when
diagnosis was established by standard histopathology only, what has been for the
most cases inexact because of the diversity that exists within these tumors, even
among those of the same grade and histologic type (Sulman et al., 2009). However,
over the past 10 years, there has been an increasing use of molecular markers, such
as methylation of the methylguanine methyltransferase (MGMT) promoter and
mutations of IDH-1 (Sulman et al., 2009), in the assessment and management of
adult malignant gliomas. Some molecular signatures are used diagnostically to help
pathologists in the classification of tumors, whereas others are used to estimate
prognosis for patients. Most important, those markers are used to predict response to
certain therapies, thereby directing clinicians to a particular treatment while avoiding
other potentially deleterious. It has also paved the way for the possibility of
personalized medicine, in which a patient’s tumor expression profile can be used to
design a treatment specific to that individual’s tumor with the greatest possibility of
response (Sulman et al., 2009). Thus, large-scale genome-wide surveys have been
used to identify new biomarkers that have been rapidly developed as diagnostic and
prognostic tools (Jansen et al., 2010).
Prognosis and therapeutic approaches depend on the type of tumor, as well as
on the location and its degree of malignancy. However, in most cases, therapy starts
with surgical removal of the tumor follow by radio and chemotherapy. Since the mid-
80s, various compounds for the treatment of gliomas have been studied, such as
cisplatin and carmustine, but none of them have proved to be effective in increasing
survival and/or improving patients outcomes (Parney and Chang, 2003). At the
present, the chemotherapeutic drugs most used are nitrosourea, etoposide, cisplatin,
vincristine and temozolomide (TMZ), being the first-line treatment with radiotherapy
and concomitant chemotherapy with TMZ. These compounds were shown to be
relatively effective used either as monotherapy or in combination with other agents
like procarbazine, lomustine, resveratrol, irinotecan and bevacizumab, among others
(Argyriou et al., 2009). In fact, Lin and colleagues described a synergistic effect of
TMZ and resveratrol, which reduced tumor volumes by inhibiting autophagy of glioma
cells after TMZ treatment, inducing apoptosis (Lin et al., 2012). This suggests that
Chapter I - Introduction
14
combined therapy could improve the efficacy of chemotherapy for brain tumors (Lin
et al., 2012).
1.5.1. Temozolomide as a chemotherapeutic agent
TMZ was developed in the 80s by the UK Cancer Research as one of a series of
novel imidazotetrazinones, and it was initially developed with the intent to treat
patients with malignant melanoma metastases in the brain. However, it also showed
activity in relapsed GBM patients, encouraging further investigation. The first in vivo
studies (phase I trial), in early 1990, confirmed the antitumoral potential of TMZ,
which also exhibited less side-effects than the conventional chemotherapeutics drugs
(Mason and Cairncross, 2005). In 1999, TMZ was approved by the FDA and,
subsequently, the European Organization for Research and Treatment of Cancer
(EORTC) and the National Cancer Institute of Canada (NCIC) demonstrated an
improved median survival, of the treatments with TMZ representing the first trials in
which such improvement was seen, since those performed with radiation therapy
(RT) in the mid-1970s (Villano et al., 2009). In 2002 and 2005 the results of phase II
and phase III trials were, respectively, published (Stupp et al., 2002, 2005), showing
the safety and efficacy of RT alone versus TMZ plus RT followed by TMZ
monotherapy (Stupp regimen or EORTC-NCIC) in patients with newly diagnosed
GBM. In the randomized phase III study, at a median follow-up of 28 months, the
median survival was 14.6 months with RT plus TMZ compared with 12.1 months for
RT alone. The 2- and 5-year survival were also improved (26,5% against 10,4%)
(Stupp et al., 2005; Villano et al., 2009). Nevertheless, the prognosis of patients are
still poor, once fewer than 3% of patients are still alive at 5 years after diagnosis
(Ohgaki, 2009).
Fig. I.4. - Chemical structure of temozolomide (TMZ) and of its metabolites. After
absorption, TMZ is spontaneously hydrolyzed at physiologic pH into the active metabolite
MTIC (5-(3-methyltriazen-1-yl)imidazol-4-carboxamid), which is quickly converted to 5-
aminoimidazole-4-carboxamide (AIC) and to the electrophilic alkylating methyldiazonium
cation that transfers a methyl group to DNA. From Villano et al. (2009).
Chapter I - Introduction
15
TMZ is a DNA alkylant agent, characterized by rapid and nearly complete oral
absorption (Friedman et al., 2000; Villano et al., 2009). After absorption, the
compound is spontaneously hydrolyzed at physiologic pH into the active metabolite
MTIC (5-(3-methyltriazen-1-yl)imidazol-4-carboxamid) (Fig. I.4). Both TMZ and MTIC
are able to cross the blood brain barrier. The active form of TMZ shows a plasma
peak concentration within 30 to 90 min following uptake, and a half-life of 2 hours.
MTIC is then quickly converted to 5-aminoimidazole-4-carboxamide (AIC) and to the
electrophilic alkylating methyldiazonium cation that transfers a methyl group to DNA.
Methylation of O6 position of guanine (O6-meG) explains the cytotoxicity of TMZ,
which leads to inhibition of proliferation or cell death, late apoptosis, senescence
(Gunther et al., 2003), autophagy, and cell cycle arrest (Fig. I.5) (Hirose et al., 2001;
Carmo et al., 2011).
Fig. I.5. – Mechanisms of activity of temozolomide (TMZ) as an enhancer therapeutics.
O6-meG (O6 position of guanine) DNA adducts, as the DNA DSB (DNA double-straind
breaks), are responsible for the cytotoxic effect of TMZ. MGMT (O6-methylguanine-DNA
methyltransferase) repairs the lesions, resulting in resistance to TMZ. When MGMT is
depleted or suppressed by methylation of the gene promoter, cytotoxicity of TMZ is
enhanced.
The O6-G-alkylation is reversed by the O6-methylguanine-DNA
methyltransferase (MGMT) and thus high levels of MGMT are thought to contribute
Chapter I - Introduction
16
to the resistance to TMZ. MGMT repairs the O6-meG lesion by transferring the
methyl group to its own cysteine residue. Methylated MGMT is then degraded. Thus,
MGMT is considered a ‘suicide’ repair protein, and new MGMT must be synthesized
in order to continue DNA repairing. Conversely, methylation of the promoter of the
MGMT gene silences the gene, and would be expected to enhance the cytotoxicity of
O6-meG lesions (Villano et al., 2009).
1.5.2. Ursodeoxycholic acid (UDCA) and its glyco- (GUDCA) and tauro-
(TUDCA) conjugated species
Ursodeoxycholic acid (UDCA), the 7β-hydroxy epimer of chenodeoxycholic acid,
is an endogenous bile acid that has been widely used for the treatment of
hepatobiliary disorders since the mid-1980s (Lazaridis et al., 2001) and is also
suggested to have a potential role in the treatment of non-liver diseases associated
with increased levels of apoptosis, since as been considered an anti-apoptotic agent
(Rodrigues and Steer, 2001). Following oral administration, UDCA is conjugated with
taurine and glycine in the liver, originating tauroursodeoxycholic acid (TUDCA) and,
mostly, glycoursodeoxycholic acid (GUDCA, 79,8%), respectively (Lazaridis et al.,
2001; Rudolph et al., 2002). Thus, GUDCA is the conjugate form of UDCA with
highest clinical relevance.
It was demonstrated that UDCA, as well as its conjugates act as cytoprotective
agents, stabilizing cell and mitochondrial membranes and preventing cytochrome c
release, consequently reducing cellular apoptosis (Guldutuna et al., 1993; Rodrigues
et al., 2000; Silva et al., 2001). Additionally, UDCA is able to suppress the production
of pro-inflammatory cytokines by inactivation of the NF-κB pathway in different cell
types (Sola et al., 2003; Joo et al., 2004; Schoemaker et al., 2004; Shah et al.,
2006). Our most recent findings showed that GUDCA suppresses the production of
the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β
in astrocytes (Brito et al., 2008). Moreover, it was recently suggested that the
cytoprotective mechanism of both UDCA and its conjugates, is mediated by a
defense against oxidative stress, pointing to antioxidant properties of the molecule
(Rodrigues et al., 2000; Lapenna et al., 2002; Serviddio et al., 2004; Perez et al.,
2006).
There are no studies regarding the effect of GUDCA on glioma cells, but it has
been described that UDCA was shown to prevent colon tumorigenesis and in
addition to its antiproliferative effect, it induces tumor growth suppression, reinforcing
its chemopreventive actions (Wali et al., 2002).
Chapter I - Introduction
17
2. Neural stem cells (NSCs)
Stem cells have been described as cells with extensive proliferative potential,
differentiation ability and self-renewal capability. NSCs can generate both neurons
and glial cells (astrocytes, oligodendrocytes and microglia). Contrary to what was
thought initially, NSCs exist also in the adult brain, playing an important role in
neuronal plasticity (Temple, 2001).
2.1. NSC in the developing and adult brain
The CNS is formed over a short time in vertebrate embryogenesis and begins as
a layer of neuroepithelial progenitors (NEPs) that rapidly form the neural tube (Merkle
and Alvarez-Buylla, 2006). The development of the CNS includes several steps, such
as the generation and differentiation of distinct cell lineages of neurons and glia,
known to be descendents of multipotent NSCs. NEPs are cells specially located in
the ventricular zone (VZ) of the neural tube, which has a great mitotic activity. Later,
these cells move to the pial (external layer) of the neural tube as they progress
through the mitotic cycle. In early embryonic phases, NEPs undergo mainly
symmetric divisions, maintaining the stemness and increasing the stem cell pool, but
then they divide asymmetrically to generate new stem cells that remain in the VZ,
and intermediate progenitors (mostly neuronal precursors, but also glial) that migrate
radially outward to its final position in the brain (Temple, 2001; Nicolis, 2007). At this
point, the proliferating precursors cells originated may differentiate into more
committed phenotypes, such as differentiated neurons or glial cells (astrocytes,
oligodendrocytes or microglia). The differentiation of the neuroepithelial stem cells
into neurons and glia proceeds in a temporal specific manner that is particular for
each region of the developing neural tube. Before differentiate and in contrast with
primary progenitors, intermediate progenitors may suffer one or more symmetrical
divisions in the subventricular zone (SVZ), above the VZ, and the subgranular zone
(SGZ) within the dentate gyrus of the hippocampus. Until birth, the SVZ increases in
size and later decreases, persisting in adult life as SVZ (Merkle and Alvarez-Buylla,
2006).
In parallel to the onset of neurogenesis, radial glia (RG) cells appear to replace
NEPs. RG cells function as stem cells for neurons (at early stages) and later for glia
(Nicolis, 2007). Like NEPs, RG cells are a transiently population in the developing
brain that divide in the VZ, still its differentiation potential is less broad than that of
NEPs (Merkle and Alvarez-Buylla, 2006).
Chapter I - Introduction
18
Within the adult mammalian brain, two major germinal regions are the SVZ
along the walls of the lateral ventricles, and the SGZ in the hippocampus. The SVZ
contains the principal concentration of dividing cells in the adult brain. This region is
formed by type B cells (or astrocyte-like neural stem cells), which are characterized
by slow division. Type B cells generate actively proliferating type C cells (transient
amplifying progenitors), which in turn produce immature neuroblasts (type A cells)
that migrate to the olfactory bulb, where they can differentiate into interneurons. Type
B cells express the astrocyte marker glial fibrillary acidic protein (GFAP), and it was
recently shown that the potential of type B cells is limited (Ihrie and Alvarez-Buylla,
2008).
The adult progenitors of the dentate gyrus are found in the SGZ of the
hippocampus, where two types of cells can be identified according to their
morphologies and expressions of molecular markers. Type 1 progenitors rarely
divide, express GFAP and SRY (sex determining region Y)-box2 (Sox2), and have a
radial process across the granular zone and ramify in the inner molecular layer. On
the other hand, type 2 progenitors divide more frequently, display short processes
and do not express GFAP. Some in vivo evidences demonstrated that type 1
progenitor cells can give rise to neuroblasts that mature to neurons and, at least
some of them, have self-renew ability and generate both astrocytes and neurons
(Miller and Gauthier-Fisher, 2009).
2.2. Applying NSC biology to glioma research: the brain tumor stem cells
(BTSCs) hypothesis
Brain tumor stem cells (BTSCs) term to often describes a subpopulation of stem
cells, with properties such as self-renewal, unlimited proliferative potential, slow rates
of division, resistance to toxic xenobiotics, high DNA repair capacity and ability to
generate partially differentiated progenies (Foreman et al., 2009) (Fig.I.6).
Divergent perspectives on the origin of a brain tumor fuel a debate that revolves
around the theory BTSCs. This theory suggests that within a tumor, there is a small
distinct cell population showing stem cell characteristics that are at the origin of the
tumor, being responsible for tumor growth and maintenance. Corroborating this
hypothesis, several groups studying brain tumors cells identified a minor population
of cells in culture that are able to self-renew, form clonogenic neurospheres and
differentiate into a broad range of cell types (Ignatova et al., 2002; Hemmati et al.,
2003; Singh et al., 2003; Galli et al., 2004; Huhn et al., 2005). These brain tumor
cells expressing the cell surface marker CD133/Prominin1 (1-35% of the cell
Chapter I - Introduction
19
population), also evidenced the stem cell marker Nestin, as well as molecular
markers associated with neural precursors such as Sox2, Bmi1, Notch, Emx2, Pax6
and Jagged1. Upon exposure to serum, these clonogenic neurospheres were able to
differentiate into a mixed population of neurons (Tuj1+), astrocytes (GFAP+) and
oligodendrocytes (PDGFR+), suggesting that they derived from a cell with
multilineage differentiation capacity – a neural stem cell (Dirks, 2008).
Later, other groups extended these findings to medulloblastoma, by evidencing
that they express NSCs proteins, including Sox2, Bmi1 and Musashi1 (Hemmati et
al., 2003). These findings confirm that (1) brain tumors contain undifferentiated
neural precursors, (2) stem-like cells possess some of the molecular features of
NSCs and (3) CD133+ cells can be used for the enrichment of tumor stem-like cells
(Singh et al., 2003; Singh et al., 2004).
Fig. I.6. – Characteristics of brain tumor stem cells (BTSCs). BTSCs are characterized by
(1) extensive self-renewal and proliferation ability, (2) expression neural stem cell (NSC)
markers, (3) capacity to generate differentiate multilineage, (4) karyotypic or genetic
alterations as well as tumorigenicity in vivo or in vitro (5).
Subsequently, studies in vitro through the injection of sorted CD133+ cells
demonstrated that they could produce orthotopic tumors in the brain of NOD/SCID
mice (an immunodeficient rodent), with common in vivo features of human GBM,
such as extensive migratory and infiltrative capacity (Galli et al., 2004), whereas
Tumor stem cells features
Self-renewal and proliferation
Neural stem cell markers
Differentiation ability
Karyotypic or genetic
alterations
Tumorigenicity in vivo and in vitro
Chapter I - Introduction
20
injection of CD133- cells failed to form tumors (Singh et al., 2004). However, recent
studies suggest a less clear distinction between the ability of CD133- and CD133+
cells to form orthotopic tumors (Bao et al., 2006; Beier et al., 2007). Although CD133
expression seems to be related to stemness, it might only indicate an intermediate,
adaptive state of a cell rather than a phenotype. It has been reported that CD133-
isolated from primary GBM were equally capable of forming orthotopic tumors as the
CD133+ population (Beier et al., 2007). These findings suggest that CD133 is not a
reliable marker for the tumorigenic capacity of stem like cells.
Currently, all stem-like glioma cells used in research are almost exclusively
derived from glioblastoma and no defined cell type for its origin has emerged, most
probably owing to the heterogeneity of the disease. Thus, the tumor brain stem cells
fraction will require further purification.
2.2.1. The origin of BTSCs
Although there is an accumulating evidence that tumors contain a subpopulation
with a tumor-initiating potencial, as described in the previous Section, the cell of
origin of BTSCs has not been determined yet. At the present, it is unclear whether a
tumor arises from NCSs, progenitor cells or differentiated cells that dedifferentiate
into a stem-like state (Fig. I.7).
Traditional neuro-oncology postulated that the differentiated glial cells were the
cells at the origin of gliomas. However, to undergo oncogenic events, mature glial
cells would have to be proliferative and it is currently accepted that most brain cells
do not divide, during adult life. Thus, numerous recent studies have been suggesting
that these tumors may arise from the transformation of NPCs or the dedifferentiation
of mature glial cells in response to genetic alterations (Llaguno et al., 2008).
The SVZ is one of the most important sources of neural stem/progenitor cells and
this region is then believed to be in the origin brain tumors. Indeed, many tumors
develop near this region (Sanai et al., 2005). In addition, corroborating the
stem/progenitor cell origin of tumors are the similarities shared by normal stem cells
and tumor stem cells, such as high mobility, extensive self-renewal and proliferation,
expression of immature profiles and association with blood vessels (Sanai et al.,
2005). Moreover, histological studies demonstrate the lack of the expression of
differentiated cell markers (Dahlstrand et al., 1992). Finally, Holland and co-workers
have found that undifferentiated cells may be more sensitive to transformation than
differentiated cells (Holland et al., 2000). Using a retroviral system, they directed the
expression of oncogenes to brain cells expressing GFAP or to cells expressing
Chapter I - Introduction
21
Nestin, and they found that malignant glial tumors arise most efficiently after
oncogene transfer to nestin-expressing cells. Taken together, these evidences point
out the involvement of immature precursors cells in the development of the tumor
phenotype, but it is not known which developmental stage, from NSC to early-
differentiated cell type lineages, are more prone to malignant transformation.
On the opposite, the existence of cells in the adult brain capable of reverting to a
less mature state in response to certain stimuli, supports the hypothesis of
dedifferentiation of mature glial cells (Canoll and Goldman, 2008). These cells were
known to dedifferentiate into transformed glia with stem cell-like properties through
retroviral transfection (Bachoo et al., 2002).
Fig. I.7. – Possible origins of brain tumor stem cells. Brain tumor stem cells (BTSCs) may
be originated from different cell types and stages. A neural stem cell (NSC) can (1)
differentiate into a neural progenitor cell or (2) suffer a transformation, originating a BTSC. In
its turn, the neural progenitor may also (3) differentiate into mature cells (such as neurons,
astrocytes or oligodendrocytes) or (4) undergo transformation into BTSCs. Notwithstanding,
mature cells may (5) de-differentiate into neural progenitor, which consequently might (4)
transform to a BTSC or (6) de-differentiate to a more immature stage (NSC), that still may
suffer transformation to a BTSC. Adapted from
http://www.igp.uu.se/Research/Cancer_and_vascular_biology/karin_forsberg_nilsson/?langua
geId=1
Thus, if we can identify the cell(s) at the origin of brain tumors, we will be better
equipped to understand which molecular alterations may lead to cancer, and how we
can target these by therapeutics or by modulators able to prevent their occurence.
The cell that is transformed may have important influences on the behaviour of the
neoplasm and therefore may also affect the patient prognosis.
5
1
2 4
3
6
DE-DIFFERENTIATION
DE-DIFFERENTIATION
Chapter I - Introduction
22
2.2.2. Therapeutic perspectives
In addition to their relatively quiescence, BTSCs are also resistant to
chemotherapy due to their enhanced capacity for DNA repair and ABC-transporter
expression (Atkinson et al., 2009). In fact it was recently described that BTSCs highly
express Mrp1 (Jin et al., 2010). Besides, these cells have a great infiltrative ability
and may activate certain survival pathways to inhibit apoptosis (Carmo et al., 2011).
Thus, new therapies targeting BTSCs may be developed if we want to prevent or
eliminate recurrent and metastatic disease.
Molecular analysis of the BTSCs population may lead to the identification of novel
pathways important for the proliferation, self-renewal and differentiation of these
cells, oppening new targets for therapy, which should be able to modify the signaling
pathways or the microenvironment favouring for their self-renewal (Singh et al., 2004;
Xie, 2009). Yet, another possibility is disrupting the interactions between BTSCs and
their niche that hopefully will slow tumor bulk malignancy progression can be slowed.
Also, the promotion of differentiation, particularly if terminally differentiated cells
types can be generated, may be another useful strategy.
Due to the infiltrative nature of BTSCs, it is difficult to specifically deliver
therapeutic agents to these cells. One option it would be to harness the potential of
normal NCSs, which exhibit strong tropism for brain tumor cells when transplanted
into the host brain. Thus, intracranial transplantation of NSCs carrying therapeutic
agent might effectively eliminate BTSCs and inhibit tumor growth (Xie, 2009).
Efflux pumps, like ABC-drug transporters, may also be targeted it a therapy of
chemotherapy and adjuvant chemosensitizers could be used with the aim of alter the
activity of some ABC transporters, leading to better clinical outcomes (Dean et al.,
2005).
Tumor cells have been found to be in a state of redox imbalance with a more
oxidizing environment, and show an increased ability to withstand oxidative stress
(Ogasawara and Zhang, 2009). Recently, several signaling pathways involved in
different cell processes, such as self-renewal, proliferation and differentiation, have
been recognized as being under redox regulation (Hernandez-Garcia et al., 2010). In
fact, some authors have already hypothesized that the highly drug resistance of
BTSCs can be due to the use of their redox regulatory mechanisms to escape the
cell death by several anticancer agents (Blum et al. 2009; Hill and Wu, 2009;
Ogasawara and Zhang, 2009; Boman and Huang, 2008; Morel et al., 2008; Tang et
al., 2008). Thus, given the significance of redox environment in BTSCs, we can
hypothesize that molecules with antioxidant potential, such as GUDCA can be used
as coadjuvants of classical chemotherapy (Szatmari et al., 2006).
Chapter I - Introduction
23
Thus, potential avenues for therapeutic intervention may require combinations of
targeted therapies against both stem-like and less tumorigenic cancer cells, as well
as directed to inhibition of resistance mechanisms in cancer stem cells. The potential
stem/progenitor glioma origin and the presence of stem-like cancer cells also paves
the way for new therapeutic avenues such the use of therapy that promotes
differentiatiom, to retard the growth of malignant astrocytomas.
24
25
CHAPTER II - OBJECTIVES
26
Chapter II - Objectives
27
The main goals of the present work are a) to identify novel cues to the cellular
pathways implicated in gliomagenesis and b) to find a successful adjuvant molecule
for TMZ therapy that may decrease chemoresistance and/or alter cell environment.
More specifically, our first aim is to identify which developmental stage, from NSC
to immature/early differentiated glia, is more susceptible to malignant transformation.
To accomplish this goal, we will use the mouse glioma cell line GL261, which will be
first characterized regarding to the expression of neural phenotypes, as well as
primary cultures of NSC, growing as neurospheres, which will be induced to
differentiate into astrocytes. Then, we will identify the neural developmental stage
more similar to glioma cells by comparing the expression of some tumor-related
markers such as multidrug resistance, angiogenesis potential, autophagy ability,
migratory and invasion capability.
In addition, we will also explore the validity of some new molecules as
coadjuvants in TMZ therapy. Thus, we will evaluate the effect of TMZ, alone in
association with GUDCA or with MK-571 (an Mrp1 inhibitor) on the viability and
proliferation of glioma cells, as well as on their cell cycle progression. We will finally,
explore the effect of GUDCA and MK-571 at the level of some migration-related
present in of glioma cells.
28
29
CHAPTER III – Materials and methods
30
Chapter III - Materials and methods
31
1. Cell cultures
1.1. GL261 mouse glioma cell line
The Gl261 was a kind gift from Dr Geza Safrany, from the National Research
Institute for Radiobiology and Radiohygiene, in Hungary. Cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom AG, Berlin, Germany)
supplemented with 38.9 mM glucose, 11 mM sodium bicarbonate, 1%
penicillin/streptomycin and 10% feral bovine serum (FBS) (Invitrogen, Carlsbad, CA,
USA), at 37ºC and 5% CO2 conditioned atmosphere during 7 days. The medium was
changed every two days and cells were passaged when the cells reached
confluence. After 3, 5 and 7 days in vitro, cells plated in coverslips were fixed with
freshly prepared 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany) during
20 min and used for immunocytochemistry assays. The ones that were in the wells
without coverslips were used for flow cytometry studies or lysed for western blot.
Growth medium was removed, centrifuged and stored at -80oC to evaluate the
release of MMPs and S100B.
1.2. Primary neurosphere culture of mouse brain cortex at E15 and induction
of astrocyte differentiation
Animal care followed the European Legislation on Protection of Animals Used for
Experimental and Scientific Purposes (EU directive L0065, 22/07/2003) in order to
ensure their well-being and minimize animals use and suffering.
Cortical neural precursors were isolated from embryonic day (E) 15. Briefly,
pregnant female mice at gestational stage E15 were euthanized by asphyxiation with
CO2. The fetuses were rapidly decapitated and after removal of meninges and white
matter, the neocortices were collected in 9 ml of Hank's Balanced Salt Solution
(HBSS, Invitrogen) and mechanically fragmented. After chemical dissociation with
trypsin-EDTA 5% (Sigma-Aldrich, St. Louis, MO, USA) and deoxyribonuclease I
bovine (DNAse I, 1 U/ml, Sigma-Aldrich), the suspension was incubated for 30 min at
37ºC, with occasional mixing. Following trypsinization, cells were washed three times
with HBSS and resuspended in 5 ml of RHB-ATM medium (Stem Cell Sciences,
Cambridge, UK). Once resuspended, cells were mechanically dissociated using a
Pasteur pipette performing around 20 passages. Approximately 1x106 cells/ml were
plated into 24-well uncoated tissue culture plates in culture medium supplemented
with growth factors (10 ng/ml, recombinant murine epidermal growth factor (EGF)
and basic fibroblast growth factor (bFGF) (PeproTech, Rocky Hill, NK, USA), to form
free-floating neurospheres, maintained at 37ºC in a humidified atmosphere of 5%
Chapter III – Materials and methods
32
CO2, during 48 h. After this period, astroglial differentiation was induced by using
10% FBS during 7 days. In neurospheres and in cells with 3 and 7 DIV under
differentiating conditions, cell lysates were collected for western blot analysis and
their growth medium was removed, centrifuged and stored at -80oC to evaluate the
release of MMPs and S100B.
2. Characterization of the mouse glioma cell line GL261
2.1. Characterization of the GL261 cells by immunocytochemistry
To characterize the glioma cell line, fixed cells were incubated in a 0.1M glycine
(Merck) solution during 10 min and then permeabilized with 0.1% Triton X-100
(Roche Diagnostics, Indianapolis, USA) solution for other 10 min. Following three
rinses with PBS, coverslips were blocked using 10% FBS in Tween 20-Tris buffered
saline (TBS-T, 0.05% Tween 20, Merck; 20 mM Tris-HCL, 500 mM NaCl, pH 7.5) for
30 min at room temperature (RT). Coverslips were incubated overnight, at 4°C, with
anti-microtubule associated protein (MAP)-2 antibody (mouse, 1:100, Millipore,
Billerica, MA, USA), anti-glial fibrillary acidic protein (GFAP) antibody (rabbit, 1:500,
Millipore); anti-nestin antibody (mouse, 1:200, Millipore), anti-glutamate transporter
(GLAST/EAAT1) antibody (mouse, 1:500, AbCam, Cambridge, UK), anti-(sex
determining region Y)-box 2 (Sox2) antibody (rabbit, 1:500, Millipore) and anti-
vimentin antibody (mouse, 1:25, Santa Cruz Biotechnology, CA, USA). Following
three rinses with TBS-T, coverslips were incubated with FITC-labelled anti-mouse
IgG (horse, 1:227, Vector Labs, Burlingame, CA, USA) and Alexa 594-labelled anti-
rabbit IgG (goat, 1:1000, Invitrogen), during 90 min at RT. After rinsed, coverslips
were incubated with Hoechst dye 33258 (Sigma-Aldrich) during 2 min for cell nuclei
staining. Following a final rinse in TBS-T and dehydration with methanol (Merck),
coverslips were mounted using DPX (BDH Prolabo, Bangkok, Thailand) and stored
at 4°C. Finally pairs of U.V. and fluorescence images of ten random microscopic
fields (original magnification: 252x) were acquired per sample. Immune-positive cells
for each cell type and total cells were counted to determine the percentage of
positive nuclei. The resultant values were presented as percentage of positive cells
for each staining.
2.2. Characterization of the GL261 cells by flow cytometry
To characterize the glioma cell line by flow cytometry, cells were trypsinized with
0,1% Trypsin-EDTA in PBS and collected. Then cells were centrifuged at 500 g for 5
min at 4ºC and washed once with Phosphate buffered saline (PBS). After that, cells
Chapter III - Materials and methods
33
were fixed with 4% PFA for 20 min in ice and centrifuged. Cells were blocked using
10% FBS TBS-T for 20 min at RT. Following centrifugation, cells were incubated
during 30 min at RT, with the antibodies mentioned in section 4.2. Cells were
incubated with FITC-labelled anti-mouse IgG (1:227) and Alexa 594-labelled anti-
rabbit IgG (1:1000), during 30 min at RT. After centrifugation, cells were rinsed once
and ressuspended in PBS. Finally, cellular suspension was plated in a 96-wells plate
and analysed by flow cytometry (Guava – Easy Cyte HT model, Millipore). Results
were expressed as percentage of positive cells for each one of the antibodies
analyzed.
3. Charaterization of tumor-related factors
3.1. MMPs activity
To compare the activity of MMPs of the GL261 cell line and with each of the
neural developmental stages, aliquots of glioma cells supernatants (3, 5 and 7 DIV),
NSC and differentiating astrocytes at 3 and 7 DIV were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography in 0.1%
gelatin/10% acrylamide (Sigma-Aldrich; Merck) gels under non-reducing conditions.
After electrophoresis, gels were washed for 1 h with 2.5% Triton X-100 (in 50 mM
Tris pH7.4; 5 mM CaCl2; 1μM ZnCl2) to remove SDS (VWR-Prolabo) and renature
the MMPs species in the gel. Then the gels were incubated in the developing buffer
(50 mM Tris pH7.4; 5 mM CaCl2; 1μM ZnCl2) overnight to induce gelatine lysis. For
enzyme activity analysis, the gels were stained with 0.5% Coomassie Brilliant Blue
R-250 (Sigma-Aldrich) and destained in 30% ethanol/10% acetic acid/H2O.
Gelatinase activity, detected as a white band on a blue background, was quantified
by computerized image analysis and normalized with total cellular protein.
3.2. S100B assay
The expression of S100B in glioma cells (at 3, 5 and 7 DIV), NSC and
differentiating astrocytes at 3 and 7 DIV was assessed by ELISA. Supernatants were
incubated with monoclonal antibody anti-S100B (1:1000, Sigma-Aldrich) in
carbonate-bicarbonate buffer (50mM, pH 9.5) per well at 4oC overnight. After three
washes with wash buffer (0.1% bovine serum albumin (BSA, Sigma-Aldrich) and
0.05% Tween(Merck)), supernants were blocked (2% BSA in PBS) for 1 h at RT. To
each sample was added 50mM Tris buffer (pH 8.6, with 0.2mM CaCl2), followed by
three washes with wash buffer. Supernants were incubated with polyclonal antibody
anti-S100 (1:5000 in 0.5% BSA with 0.2 mM Cacl2 in PBS, Dako, Dernmark, A/S) for
30 min at 37oC and, then, incubated with antibody anti-rabbit peroxidase conjugated
Chapter III – Materials and methods
34
(1:5000 in 0.5% BSA in PBS, Santa Cruz Biotechnology) under same conditions.
Following washes with both wash buffer and PBS, it was added substract solution
(Sigma Fast OPD in H2O, Sigma-Aldrich) for 30 min at RT and under light protecting.
At last, absorbance was measured at 450 nm.
3.3. Expression of tumor-associated factors
To compare some tumor-associated factors of the GL261 cell line with the
different neural developmental stages, total cell extracts of both glioma cells (3, 5
and 7 DIV), NSC and differentiating astrocytes at 3 and 7 DIV, were obtained by
lysing cells in ice-cold Cell Lysis Buffer (Cell Signaling, Beverly, MA, USA) plus 1 mM
phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich) for 10 min, on ice, followed by
sonication. The lysate was centrifuged at 14 000 g for 10 min, at 4ºC, and the
supernatants were collected and stored at -80ºC. Protein concentrations were
determined using the Bradford assay. Equal amounts of protein were subjected to
SDS-PAGE and transferred to a nitrocellulose membrane (Amersham Biosciences,
Piscataway, NJ, USA). After transfer, blotted membranes were blocked for 1 hour at
RT with 4% milk in TBS-T (in case of Mrp1 expression evaluation) and in 5% BSA
(Sigma-Aldrich) in TBS-T (for determination of VEGF, β-actin and LC3) and
incubated overnight at 4ºC with anti- multidrug resistance protein 1 (Mrp1)-A23
antibody (1:750, Sigma-Aldrich), anti-vascular endothelial growth factor (VEGF)
antibody (1:200, Santa Cruz Biotechnology), anti-β-actin antibody (1:5000, Sigma-
Aldrich) and anti-protein light chain 3 (LC3) antibody (1:2000, Cell Signaling) in
respective blocking solution. After washing with TBS-T, the membranes were
incubated with secondary antibody anti-rabbit (horse, 1:5000, Santa Cruz
Biotechnology) or anti-mouse (goat, 1: 5000, Amersham Biosciences), as
appropriate, in blocking buffer for 1 h, at RT. After washing membranes with TBS-T,
chemiluminescent detection was performed by LumiGLO® (Cell Signaling) and
bands were visualized by autoradiography with Hyperfilm ECL. The relative
intensities of protein bands were analyzed using the Quantity one® 1-D
densitometric analysis software (Bio-Rad, Hercules, CA, USA).
4. Cell treatments
Glioma cells were first treated (or untreated, control) with TMZ (50, 100 and 250
µM, Sigma-Aldrich) during 24, 48 and 72 h. After incubation, cell viability was
evaluated in order to ascertain the most efficient TMZ incubation conditions to be
subsequently used. After this first trial, glioma cells were then incubated with TMZ
alone or in the presence of MK-571 (25 µM, Sigma-Aldrich), or GUDCA (50 µM,
Chapter III - Materials and methods
35
Calbiochem Darmstadt, Germany) at selected exposure conditions. After incubation,
it was determined the cell viability, proliferation, cell cycle progression and cell death
by apoptosis. The success of GUDCA or MK-571 co-incubation was evaluated by
comparing the results with those obtained with TMZ alone.
Glioma cells with 3, 5 and 7 DIV were also incubated with MK-571 (25 µM), or
GUDCA (50 µM) at the previously selected exposure time, to explore the effect of
these molecules on some migration-related factors of glioma cells, such as CXCR4
expression.
4.1. Cell viability
Cell viability was determined by evaluating [3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium] (MTS) reduction in the
presence of phenazine methosulfate (PMS), which forms a formazan product that is
released to the culture medium, with an absorbance maximum at 490 nm.
A combined MTS/PMS solution (1:20, with stock solution at 2 mg/ml and at 0.92
mg/ml, respectively) was freshly prepared and after the cell treatment, supernatants
were removed and cells incubated for 45 min, at 37°C, in a dilution of 1:10 in culture
medium. At the end of incubation, the absorbance of the medium was read at 490
nm using an ELISA plate reader.
4.2. Cell cycle progression
For determination of cell cycle progression, the cells were analyzed by flow
cytometry. At the end of incubation period, cells were collected, washed with PBS,
centrifuged at 500 g for 10 min, and then the pellet was resuspended in a fixative
solution with glycine:ethanol (3:7, volume/volume) solution for 30 min at 4ºC. After
centrifugation at 500 g for 10 min, cells were washed with PBS and the pellet
resuspended and incubated for 10 min in the dark, at RT, in a solution of PBS
containing 10μL/mL propidium iodide (PI, Invitrogen, Paisley, UK) and 10μL/mL
RNAse. The PI fluorescence was measured on a FACScan flow cytometer (BD
FACSCaliburTM) and the data were gated to exclude cell debris and aggregates.
4.3. Expression of CXCR4
To evaluate CXCR4 expression, total cell extracts of GL261 cells (3, 5 and 7 DIV)
were obtained by lysing cells in ice-cold Cell Lysis Buffer plus 1 mM
phenylmethylsulfonyl fluoride (PMSF) for 10 min, on ice, followed by sonication. The
lysate was centrifuged at 14 000 g for 10 min, at 4ºC, and the supernatants were
collected and stored at -80ºC. Protein concentrations were determined using the
Chapter III – Materials and methods
36
Bradford assay. Equal amounts of protein were subjected to SDS-PAGE and
transferred to a nitrocellulose membrane. After transfer, blotted membranes were
blocked for 1 h at RT in 5% milk TBS-T and incubated overnight at 4ºC with anti-
CXCR4 antibody (rabbit, 1:1000, AbCam), and anti-β-actin antibody (1:5000) in
respective blocking solution. After washing with TBS-T, the membranes were
incubated with secondary antibody anti-rabbit (1:5000) or anti-mouse (1: 5000), as
appropriate, in blocking buffer for 1 h, at RT. After washing membranes with TBS-T,
chemiluminescent detection was performed by LumiGLO® and bands were
visualized by autoradiography with Hyperfilm ECL. The relative intensities of protein
bands were analyzed using the Quantity one® 1-D densitometric analysis software
(Bio-Rad, Hercules, CA, USA).
37
Chapter IV – Results and discussion
38
Chapter IV - Results and discussion
39
1. Characterization of the mouse glioma cell line GL261
In this Chapter of Results we have decided to continuously evaluate and
moderately discuss the significance of the values obtained to better understand the
relevance of the values achieved in each point, due to the novelty of the approach
we have programmed to follow.
The application of suitable experimental models to glioma research, which
ideally should harbor key features of the human disease, is necessary for the
identification of more specific targets and development of novel and target-directed
therapies. One of the most widely used for preclinical and translational research is
glioma 261 (GL261) cell line (Wu et al., 2008). These cells carry point mutations in
the K-ras and p53 genes (Szatmari et al., 2006) and exhibit, as other glioma cell
lines, populations of cells that have characteristics of cancer stem cells, such as the
CD133+ cells (Wu et al., 2008), as well as a sub-population of cells more sensitive to
ATP (Tamajusuku et al., 2010). The choice of the GL261 cell line, instead of the
other currently used cell line C6 derived from rat glioma cells and representing
astrocyte-like cells (Swarnkar et al., 2012), was based on data evidencing that C6
gliomas are slightly invasive and only induce moderate vascular alterations, whereas
GL261 tumors dramatically alter the brain vessels in the glioma region (Doblas et al.,
2010), a property that we were interested to explore in the present work. Moreover,
GL261 cells were recently considered, between several tested rodent glioma models,
the one showing the greatest alterations in glioma metabolites (e.g. glutamate,
lactate, total choline and creatine, glutamine-, aspartate, guanosine, mobile lipids
and macromolecules, among others) (Doblas et al., 2012).
To assess the different stages of differentiation and the several cell types that
may constitute the GL261 cell line, we started by using specific antibodies against
proteins that are characteristic of undifferentiated and differentiated cells, at three
different time points – 3 days in vitro, 5DIV and 7DIV. In order to characterize and
evaluate the content in undifferentiated proliferating cells, it was used antibodies
against Nestin and Sox2. Vimentin was used to stain early astrocyte progenitors and
antibodies against βIII-Tubulin and MAP2 to identify neuronal cells. The astrocytic
population was evaluated through the use of antibodies specific for GFAP and
GLAST.
Initially, the characterization was performed by using immunocytochemistry
(Fig.IV.1A.). We have observed that although all glioma cells were positive to the
antibodies tested, it can be observed a different staining pattern. In fact, it seems that
the markers more related to differentiated cells, such as GFAP and MAP2, were
particularly evident in the cytoplasm and thus we can clearly visualize cell
Chapter IV – Results and discussion
40
ramifications, while GLAST is located near nuclei. The markers more related to
undifferentiation, such as Nestin and Vimentin, stained cytoskeleton, whereas Sox2
labeled the perinuclear zone. However, this method enabled us to more accurately
quantify the different phenotypes of GL261 cells along the time in culture, since all
the cells were labeled.
Thus, the characterization proceeded by using the flow cytometry. This method is
often used, not only for being a faster one, but also because of its higher specificity.
The evaluations were optimized and the data obtained by flow cytometry confirmed
that, in fact, the cellular composition of the GL261 cell line was not the same along
the culture time window.
As shown in Fig. IV.1B, and despite only one assay has been performed, it can
be observed that the expression of Vimentin increased 15,2% from 3 to 5 DIV and
then remained constant until 7DIV, whereas the expression of both Sox2 and βIII-
Tubulin decreased from 3 to 7 DIV, the decrease was more marked for Sox2 (40%)
than for βIII-tubulin (23%). Because Sox2 is a glioma stem cell marker (Allen et al.,
2012) data indicate that the stem cell representation in the GL261 cell line declines
significantly and continuously (Fig. IV.1B) along the time in culture. Being Sox2 an
undifferentiated cell marker, we can speculate that the early GL261 3DIV, may
correspond to a more stem cell-like population and thus, with higher proliferation
ability. In fact, undifferentiated phenotypes, this is, stemness phenotypes, have been
associated to self-renewal capacity with implications toward possible roles in brain
tumorigenesis (Shiras et al., 2003; Gangemi et al., 2009). Gangemi et al have
denoted that Sox2 silencing in GBM cancer stem cells drive to proliferation inhibition
and the loss of the tumorigenicity in immunodeficient mice, demonstrating the
fundamental role for maintenance of the self-renewal capacity of neural stem cells
when they have acquired cancer properties. Therefore, it is speculated that SOX2, or
its immediate downstream effectors, would then be an ideal target for glioblastoma
therapy.
βIII-tubulin is a neuronal differentiation marker aberrantly expressed in astrocytic
gliomas (Katsetos et al., 2003) and linked to malignant changes in glial cells
(Katsetos et al., 2007). Its overexpression has been related with chemoresistance
(Zheng et al., 2012) and accumulation of βIII-tubulin was observed around the G2/M
stage of the cell cycle of tumor cells (Shibazaki et al., 2012). The increased
expression of βIII-tubulin in GL261 cells asserts, thus, a link between its aberrant
expression and a disruption of microtubule dynamics usually observed during the
transformation of a low-grade to a high-grade glioblastoma (Katsetos et al., 2011).
Therefore, we may assume that GL261 even at 7DIV differentiation still have glioma
Chapter IV - Results and discussion
41
tumorigenesis, tumor progression and malignant transformation characteristics of
glioblastoma multiform (Katsetos et al., 2009).
Vimentin, a mesenchymal marker (Ma et al., 2012), is a primordial component of
the cytoskeleton and the nuclear envelope (Wang et al., 2010) that has been used as
a molecular marker for glioblastoma multiform and astrocytoma (Yang et al., 1994;
Mennel and Lell, 2005). The increase in Vimentin at both 5 and 7DIV (Fig. IV.1B)
may result from increased cell migration abilities as its up-regulation is associated
with tumor invasiveness (Jan et al., 2010; Thakkar et al., 2011).
GFAP is widely expressed in astroglial cells, neural stem cells, astroglial tumours,
such as glioblastoma. It is consider a diagnostic marker of glioblastoma multiforme
because GFAP presence is associated with more aggressive and invasive potentials
(Jung et al., 2007). These authors observed that the most uniform GFAP staining
was in well-differentiated grade II astrocytomas. We found that the expression of
GFAP was almost the same along the time in culture (~70% of positive cells), with a
10% decrease at 7DIV. Once GFAP expression is related to the differentiation status
of astrocytes (Jung et al., 2007) we can hypothesize that cell proliferation in our case
is not so much elevated. Interestingly, many high-grade gliomas also seem to lose
GFAP expression (Jacque et al., 1978; van der Meulen et al., 1978; Jacque et al.,
1979; Velasco et al., 1980; Tascos et al., 1982) (Rutka et al., 1997). In addition,
GFAP-negative cells proliferate more rapidly than GFAP-positive cells in the same
tumor (Hara et al., 1991; Kajiwara et al., 1992). These in vivo findings allow
demonstrate that the loss of GFAP expression could represent secondary loss of a
differentiation marker or alternatively, it could be a step in tumor development
(Wilhelmsson et al., 2003). Thus, we may speculate that GL261 at 7DIV have
increased its aggressiveness and invasive potentials in comparison to 3DIV cells.
Finally, GLAST and MAP2 are expressed unevenly, with increased levels at 5
DIV. GLAST is an astroglial glutamate transporter that was shown to be present in
glioma cells (Baber and Haghighat, 2010) at similar levels to those of astrocytes,
although its mislocation was noticed as an intrinsic feature of glioma cells (Ye et al.,
1999). Variations in the Wnt-1 oncogene expression (Palos et al., 1999; Jimenez et
al., 2003) may be in the origin of the observed GLAST fluctuation between GL261
from 3 to 7DIV, and deserve to be evaluated in the future.
MAP2 is another early neuronal marker that was shown to be also present in
glioma cell lines and biopsies (Yan et al., 2011). In fact, MAP2 expression was
demonstrated to occur transiently in migrating immature glial cells and indicated as
corroborating the glial origin of the gliomas (Blumcke et al., 2001).
Chapter IV – Results and discussion
42
A)
3DIV 5DIV 7DIV G
FA
P
GL
AS
T
Vim
en
tin
So
x2
Ne
sti
n
MA
P2
Chapter IV - Results and discussion
43
B)
Fig. IV.1. Characterization of the glioma cell line GL261. Glioma cells were maintained in DMEM
supplemented with 1% PenStrep and 10% FBS as previously described in Methods. Cells were fixed at
3, 5 and 7 days in vitro and processed for immunocytochemistry (A), where nuclei were stained with
Hoechst dye (blue), or toflow cytometry analysis (B). Representative images (A) and data obtained from
a single independent experiment (B). GFAP, glial fibrillary acidic protein, GLAST, glutamate aspartate
transporter, MAP2, microtubule-associated protein 2. Scale bar: 20 μm.
Overall, these results indicate that the sub-fractions of cells that constitute the
GL261 cell line and attest the tumor heterogeneity feature (Deleyrolle et al., 2012)
change in accordance with the time of cells in culture. Also, indicate that
independently of the time in culture these cells exhibit a primary tumor phenotype
and highlight their value to explore the origin of gliomas and for preclinical modeling
of novel anti-glioblastoma therapeutic agents. However, taking in account the results
of a sole experiment, careful should be taken in the appreciation of the results just
presented, and new series should be undertaken in the near future to validate them.
In the next section, we decided to compare some important biological glioma like
properties of GL261 cells at the 3 different culture temporal windows with 3 steps of
neural precursors cell differentiation.
2. Characterization of common features between GL261 glioma cells and
differentiating astrocytes from neural stem cells
NSCs, due to their longevity, self-renewal, high motility and sustained
proliferative capacity, are believed to be in the origin of the glioma (Ignatova et al.,
2002; Sanai et al., 2005) and their pluripotency the cause of the cellular diversity of
the tumor (Tan et al., 2006; Louis et al., 2007). Therefore, it is plausible that in a
particular point of NSC differentiation they are at an increased risk for malignant
transformation. This risk may be associated with a phenotype that will most
resemble equivalent one in glioma cells. To explore this resemblance, we
0
20
40
60
80
100
120
GL261 3DIV GL261 5DIV GL261 7DIV
Po
siti
ve
ce
lls
(%)
GFAP GLAST Vimentin Sox2 MAP2 βIII-Tubulin
Chapter IV – Results and discussion
44
characterized and compared some features associated to brain tumors, in glioma
cells, as well as on primary cultures of NSC, growing as neurospheres, which were
induced to differentiate into astrocytes. This way, we propose to identify the neural
developmental stage more similar to glioma cells by comparing the expression of
some tumor-related factors such as multidrug resistance, angiogenesis potential,
autophagy ability and invasion capability.
2.1. Invasion ability
One of the most important characteristics of tumor cells is their ability to invade
the surrounding tissue. This feature is associated to the presence of proteins, like
MMPs and S100B.
Interestingly, the activity of MMPs, both MMP-2 and MMP-9, decreased along the
different time points in GL261, reaching very low values at 7DIV (0.3- and 0.2- fold
vs. GL261 3DIV p<0.01, respectively). The activity of these gelatinases increased
from neurospheres to 3 and 7 DIV, thus approaching the levels observed at GL261
3DIV. Conversely, the value obtained in neurospheres was close from the one
observed in GL261 at 5 and 7DIV. Thus, it seems that Sox2 and βIII-tubulin, which
were more expressed at GL261 at 3DIV may be related with an increased MMP
expression and mobility. Intriguingly, in a recent study Oppel et al. (Oppel et al.,
2011) reported that the knockdown of Sox2 impaired the invasive proteolysis-
dependent migration of glioma cells also reducing the expression level of pro-MMP1
and pro-MMP2, and that Sox2 plays a role in the maintenance of a less differentiated
glioma cell phenotype. In addition, silencing of MMP-2 evidenced to reduce stem cell
migration and tropism towards the tumor cells (Bhoopathi et al., 2011). Further, the
expression of active MMP-2 and MMP-9 was indicated to enhance with the growth of
malignant gliomas (Zhao et al., 2007) and their down-regulation evidenced to reduce
glioma stem cell proliferation (Reddy et al., 2011) and invasion (Annabi et al., 2008;
Silveira Correa et al., 2010). To emphasize that βIII-tubulin positive immunoreactivity,
although less documented, was also related with a cell active migration (Katsetos et
al., 1998). Finally, based on these results we can speculate that GL261 3DIV and 3
and 7DIV differentiating astrocytes are those with most invasive ability.
The release of S100B into the extracellular medium revealed to increase with the
time in culture, with the highest values in both GL261 and differentiating astrocytes at
7DIV (Fig. IV.3). S100 proteins are known to be involved in proliferation,
differentiation and migration/invasion among other aspects (Donato et al., 2012). A
recent study demonstrated that the transfection of S100B promotes cell invasion and
migration and can be related with the development of brain metastasis (Pang et al.,
Chapter IV - Results and discussion
45
** ** **
§ #
§ #
0,0
0,5
1,0
1,5
2,0
GL3DIV
GL5DIV
GL7DIV
NS 3 DIV 7 DIV
MM
P2
act
ivit
y
(fo
ld c
ha
ng
e)
Differentiating astrocytes
** ## **
§ **
§§ ## §§
#
0,0
0,5
1,0
1,5
2,0
GL3DIV
GL5DIV
GL7DIV
NS 3 DIV 7 DIV
MM
P9
act
ivit
y
(fo
ld c
ha
ng
e)
Differentiating astrocytes
2012). However, the majority of the data published were related with the invasion
property of lung cancer cells in the brain (Hu et al., 2010; Jiang et al., 2011).
A)
B)
Fig. IV.2. Metalloproteinase (MMP)-2 and MMP-9 activities in GL261 glioma cells at 3, 5 and 7
days in vitro and in differentiating astrocytes from neurospheres (NS) during 3 and 7 DIV. Cells
were cultured as indicated in methods. Cell supernatants were collected for quantification of MMP
activity. A) MMP-2 and MMP-9 were identified by their apparent molecular mass of 72 and 92 kDa,
respectively. Representative results from one experiment are shown. B) Graph bars represent the
intensity of the bands that were quantified by scanning densitometry, standardized to respective protein
quantification and expressed as mean ± SEM from at least three independent experiments. Results are
and presented as fold change compared to GL261 3DIV (considered as 1). Data obtained from at least
three independent experiments. **p<0.01 and *p<0.05 vs. GL261 3DIV; ##
p<0.01 and #p<0.05 vs..
GL261 5DIV; §§
p<0.01 and §
p<0.05 vs. GL261 7DIV.
Concentrations in glioma cells (~0.5 to 7 μM, from 3DIV to 7DIV) are several
times higher than in neurospheres (~0.1 nM) or differentiating astrocytes (~0.2 µM),
thus indicating a substantial difference between both types of cells, that surely
deserves further investigation. Correspondingly, it is described that at nanomolar
concentrations, as the ones observed in neurospheres, S100B exerts neurotrophic
properties for normal brain development (Rothermundt et al., 2003). The increase of
S100B during the astrocyte differentiation process may be related to the fact that
S100B expression also characterizes a terminal maturation stage of cortical
astrocytes, since astrocytes do express S100B in the mature nervous system (Brozzi
et al., 2009). Contrastingly, at micromolar concentrations, similar to the ones found
GL 3DIV GL 5DIV GL 7DIV NS 3 DIV 7 DIV
MMP9
MMP2
92 kDa
72 kDa
Chapter IV – Results and discussion
46
in our glioma cells, S100B could be participate in the pathophysiology of some brain
diseases, including brain cancer. These concentrations, revealed to increase cellular
proliferation (Leclerc et al., 2007) and Brozzi et al. (Brozzi et al., 2009) suggested
that S100B may contribute to reduce the differentiation potential of cells of the
astrocytic lineage, beyond the contribution to enhance migration capability, suggests
that this protein might contribute to maintaining a neoplastic, invasive phenotype. In
fact, Vos et al related high levels of S100B with shorter survival in a relatively high
proportion of patients with GBM (Vos et al., 2004).
Fig. IV.3. S100B release from GL261 glioma cells at 3, 5 and 7 days in vitro and in differentiating
astrocytes from neurospheres (NS) during 3 and 7 DIV. Cells were cultured as indicated in methods.
The conditioned media was collected, and S100B released into the medium was determined by ELISA,
with monoclonal antibody anti-S100B. Quantitative analysis of S100B release was expressed as fold
increase vs. GL261 3DIV (considered as 1); **p<0.01 and **p<0.05 vs. the GL261 3DIV; #p<0.05 vs.
GL261 5DIV; §p<0.05 vs. GL261 7DIV.
Overall, when comparing these developmental phenotypes to glioma cells, the
most close to GL261 3DIV are the differentiating astrocytes, regardless the
concentration values be significantly different, as it was also observed for MMPs.
2.2. Angiogenesis
Glioblastoma is characterized by its capacity to induce neovascularization, driving
continued tumor growth, due to its high content in VEGF and autocrine signaling (Lee
et al., 2011). As previously referred, angiogenesis might be triggered and enhanced
by the release of VEGF, which is a protein regulated by hypoxia through HIF-1. In
Fig. IV.4, a decrease in VEGF expression during the time in culture was noticed in
both glioma cells and astrocytes differentiated from neurospheres. In the first case,
there was a 50% reduction from GL261 at 3 DIV to 5 DIV (p<0.01). Similar
expression was also observed in neurospheres and differentiating astrocytes at 3
# *
§ **
§ **
§ **
0
5
10
15
20
GL 3DIV GL 5DIV GL 7DIV NS 3 DIV 7 DIV
S1
00
B r
ele
ase
(f
old
ch
an
ge
)
Differentiating astrocytes
Chapter IV - Results and discussion
47
DIV, followed by a decrease at 7 DIV (i.e. a change from 0.6- to 0.3-fold, p<0.01).
However, it should be emphasized that these results correspond to two experiments
(n=2) and thus, further data should be acquired in order to corroborate these
observations, also using GL261 cells with 7 DIV.
A)
B)
Fig. IV.4. VEGF expression in GL261 glioma cells at 3, 5 and 7 days in vitro and in differentiating
astrocytes from neurospheres (NS) during 3 and 7 DIV. Cells were cultured as indicated in methods.
Total cell lysates were subjected to SDS-PAGE followed by Western blotting with antibody specific for
VEGF. A) Representative results from one experiment are shown. B) Graph bars represent the intensity
of the bands, which was quantified by scanning densitometry, standardized with respect to β-actin
protein and expressed as mean ± SEM fold change compared to glioma cells. The values indicate the
fold change obtained when compared with GL261 at 3DIV (considered as 1). **p<0.01 vs. GL261 3DIV.
Interestingly, it was recently observed by immunohistochemistry that the
percentages of tumors expressing VEGF (96%) and MMP-9 (75%) are in the glioma
high-grade group, exhibiting higher levels than in the low-grade group (67% and
24%, respectively) and correlated to the invasion of glioma (Liu et al., 2011). Our
results evidence the higher malignancy of GL261 at 3DIV with equivalent increased
values of MMP-9 (see Fig. IV.2). Moreover, it deserves to be noted that both
neurospheres and differentiating astrocytes at 3 DIV still contain elevated levels of
VEGF and similar to those presented by GL261 cells at 5 DIV, thus evidencing a
close affinity. Elevated expression of VEGF in NSCs has been documented and it
has been unveiled an intrinsic relationship between angiogenesis and NSC, where
** **
**
0,00
0,20
0,40
0,60
0,80
1,00
GL 3DIV GL 5DIV NS 3 DIV 7 DIV
VE
GF
ex
pre
ssio
n
(fo
ld c
ha
ng
e)
Differentiating astrocytes
GL 3DIV GL 5DIV NS 3 DIV 7 DIV
VEGF
β-actin
42 kDa
42 kDa
Chapter IV – Results and discussion
48
up-regulation of VEGF lead to the increase of Nestin positive cells (Mani et al 2005,
Sun et al 210). Moreover, inhibition of the VEGF signaling was shown to reduce the
migration and to induce differentiation (Kaus et al., 2010; Joo et al., 2012).
2.3. Multidrug resistance
The drug resistance of tumors is one of the main causes to treatment fail and to
the progress of the disease over the years. There are diverse reasons why tumor
cells can resist to chemotherapeutic drugs, and the transporter Mrp1 is one of them.
Mrp1 revealed to be significantly up-regulated in cancer stem-like cells (Jin et al.,
2008) and in CD133+ human brain glioma stem cells (Bi et al., 2007), besides being
more expressed in the high grade glioma (Calatozzolo et al., 2012), reason why it
has been suggested that chemosensitization of cells with Mrp1 inhibitors may favor
the treatment of gliomas (Peignan et al., 2011).
A)
B)
Fig. IV.5. Mrp1 expression in GL261 glioma cells at 3, 5 and 7 days in vitro and in differentiating
astrocytes from neurospheres (NS) during 3 and 7 DIV.). Cells were cultured as indicated in
methods. Total cell lysates were subjected to SDS-PAGE followed by Western blotting with antibody
specific for Mrp1. A) Representative results from one experiment are shown. B) Graph bars represent
the intensity of the bands quantified by scanning densitometry, standardized with respect to β-actin
protein and expressed as mean ± SEM fold change compared to glioma cells, from at least 3
experiments. The values indicate the fold change obtained when compared with GL261 at 3DIV (taken
as 1). **p<0.01 vs. GL261 3DIV; ##
p<0.01 and #p<0.05 vs. GL261 5DIV.
Differentiating astrocytes
**
## **
**
## **
#
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
GL 3DIV GL 5DIV GL 7DIV NS 3 DIV 7 DIV
Mrp
1 e
xp
ress
ion
(f
old
ch
an
ge
)
Mrp1
β-actin
GL 3DIV GL 5DIV GL 7DIV NS 3 DIV 7 DIV
190 kDa
42 kDa
Chapter IV - Results and discussion
49
The results of the analysis of Mrp1 expression depicted in Fig. IV.5. show that
despite the existence of a 1.3-fold increase from day 3 to day 5 of glioma cells
(p<0.01), there is an unexpected marked decrease from 5 to 7 DIV (0.2-fold vs.
GL261 3DIV, p<0.01). Moreover, results obtained in neurospheres were those
presenting a higher similarity to GL261 cells at both 3 and 5 DIV. However, we can
also consider that a matched correspondence still exist between GL261 at 7 DIV and
astrocytes in differentiation from NS, although with significant lower levels of Mrp1
(decline to 0.3- and 0.5-fold) that the first group above mentioned.
Overall, it seems that the increased values of Mrp1 are associated with
greater cell proliferation ability, as other ongoing studies also suggest. Further
studies on Mrp1 should include the evaluation of Mrp1 activity (Lee et al., 2012), in
order to investigate if an increased expression of this protein is related with a higher
ability to make the efflux of some drugs, and thus, to an increased chemoresistance.
As this multidrug resistance phenomenon is considered to be the major barrier to
patient survival, a chemotherapeutic scheme that include Mrp1 modulators, may be
imperative to overcome the conventional drug resistance in patients with relapsed
GBM.
2.4. Autophagy
Autophagy is a process that promotes sequester and degradation of bulk
cytosolic proteins and damaged organelles by the lysosome. It has been shown that
some drugs used in chemotherapy may activate autophagy instead of apoptosis in
malignant glioma cells (Kanzawa et al., 2004). Therefore, the expression of LC3, one
of the autophagosome-membrane proteins, was analyzed. Autophagic activity can be
analyzed by Western blot through by the ratio of lapidated LC3-II that studs the inner
and outer autophagosoma membrane to unmodified LC3-I.
The results depicted in Fig. IV.6 show that LC3 is constitutively expressed in all
cell types, GL261, NS and differentiating astrocytes. Due to limitation of time and
some identification band problems only data from one experiment are shown.
Nevertheless, there is indication that LC3 lipidation increase along differentiation
of astrocytes from neurospheres, which showed the lowest levels. Autophagic activity
in differentiating astrocytes evidenced a 1.3-fold increase when compared to GL261
5DIV. This is not without precedent once the autophagic activity in glioma
stem/progenitor cells was shown to be significantly lower than that in neural
stem/progenitor cells. However, the autophagic activity markedly increased if glioma
stem/progenitor cells are induced to differentiate by fetal calf serum (Zhao et al.,
2010).
Chapter IV – Results and discussion
50
A)
B)
Fig. IV.6. LC3II/I expression in glioma cells at 5 days in vitro and in differentiating astrocytes
from neurospheres (NS) during 3 and 7 DIV. Cells were cultured as indicated in methods. Total cell
lysates were analyzed by Western blotting with antibody specific for LC3 and then quantified and treated
as described before. A) Representative results from one experiment are shown. B) Graph bars
represent the values indicating the density proportion of each protein compared with GL261 5DIV (taken
as 1). Results are from one experiment.
Nevertheless further data should be obtained, as these results were acquired
from one single experiment, and also it should be used the missing GL261 at 3 and 7
DIV, in order to look for the effect of the time in culture on this cell property, based on
the findings above indicated. Enhanced LC3-II/LC3-I expression in differentiating
astrocytes from neurospheres is in agreement with other studies suggesting that
neural stem/progenitor cells activate autophagy to fulfill their high energy demands
(Vazquez et al., 2012). Autophagy was similarly indicated to play an essential role in
the regulation of self-renewal, differentiation, tumorigenic potential and
radiosensitization of glioma-initiating cells. Moreover, it is suggested that induction of
autophagy promotes the differentiation of these cells and their susceptibily to
radiotherapy (Zhuang et al., 2011; Palumbo et al., 2012; Teres et al., 2012).
However, it deserves to be noted that CD133+ glioblastoma cells, considered as a
small fraction of cells with features of primitive neural progenitor cells and tumor-
initiating function in brain tumors, show defective autophagy, which probably relates
0
0,5
1
1,5
GL 5DIV NS 3 DIV 7 DIV
LC
3-I
I/I
ex
pre
ssio
n
(fo
ld c
ha
ng
e)
Differentiating astrocytes
GL 5DIV NS 3 DIV 7 DIV
LC3-I
LC3-II
β-actin
17 kDa
15 kDa
42 kDa
Chapter IV - Results and discussion
51
with their resistance to TMZ (Fu et al., 2009). Thus, we may considerer that this
important glioma subpopulation should be a target to combined therapy using TMZ
and inducers of this signaling pathway, in order to improve the survival of patients
with glioma. Nevertheless, caution should also be undertaken as other studies have
revealed that the inhibition of autophagy favors TMZ-induced apoptosis in glioma
cells, and that agents targeting mitochondria or endoplasmic reticulum may be
potential anticancer strategies (Lin et al., 2012). This is a very controversial subject
deserving clarification.
3. Effects of a combined anticancer strategy on GL261 cell viability and
cell cycle
Despite the new insights into the molecular pathogenesis of glioblastoma, several
aspects are not full elucidated and treatments fail to cure the majority of patients.
With standard therapy, which consists of surgical resection with concomitant TMZ in
addition to radiotherapy followed by adjuvant TMZ, the median duration of survival is
12-14 months. Therefore, therapeutic schemes clearly deserve to be improved, and
novel molecular targets and inhibitory agents has become a focus of research for
glioblastoma treatment. Thus, the second main objective of this thesis was to find a
successful adjuvant molecule for TMZ therapy that would enhance TMZ therapeutics
potential. For that we started to evaluate the effect of TMZ in GL261 cell line, at the
cell viability and cell cycle levels, followed by the analysis of conjoint association
effects of some other molecules, such as GUDCA and an Mrp1 inhibitor, the MK-571.
3.1. Effect of TMZ on glioma cells viability
GL261 glioma cells were treated (or untreated, control) with TMZ (50, 100 and
250 µM) during 24, 48 and 72 h. After incubation, cell viability was evaluated by the
MTS assay, in order to determine the most efficient TMZ incubation conditions to be
subsequently used in the following studies. We have observed that TMZ treatment
induced a significant decrease in glioma cells viability, which occurred in a dose- and
time-dependent manner (Fig. IV.7.). Thus, the effect of TMZ was particularly evident
at the highest TMZ concentration, with an incubation period of 72 h. At these
conditions, TMZ 250 µM was able to induce a 40% decrease on cell viability (p<0.05)
as compared to the respective control cells.
Chapter IV – Results and discussion
52
Fig. IV.7. Effect of temozolomide (TMZ) addition in glioma cells viability. Glioma cells were
incubated with crescent concentrations of TMZ (50, 100 e 250 μM) during three different incubation
periods (24, 48 and 72 h). Cell proliferation was determined by evaluating [3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) reduction in the presence of
phenazine methosulfate (PMS), and absorbance of the medium was then red at 490 nm. Data are
expressed as percentage ± SEM of the value obtained for the control at each incubation period.
**p<0.01 and *p<0.05 vs. control.
Based on the results achieved we selected to the following assays a TMZ
concentration of 250 µM, and the lowest and the highest incubation periods (24 and
72 h), to easily compare the effects produced by the combined therapy.
3.2. Effect of TMZ, GUDCA and TMZ+GUDCA on glioma cells viability and
cell cycle
It was recently demonstrated that TMZ-treated glioma cells undergo ROS/ERK-
mediated apoptosis and autophagy, during which autophagy serves to protect glioma
cells from TMZ-induced apoptosis. Furthermore, resveratrol, an antioxidant molecule,
augments the effect of TMZ by reducing autophagy and increasing apoptosis both in
vitro and in vivo (Lin et al., 2012). Overall, those studies suggested that the
administration of resveratrol combined with TMZ could be a potential treatment
strategy for patients with brain tumors. Therefore, we sought that was also very
interesting to explore if the antioxidant GUDCA, a molecule with anti-proliferative,
anti-apoptotic and anti-inflammatory properties (Akare et al., 2006), which has
already been shown to prevent colon tumorigenesis (Wali et al., 2002), could also be
a successful adjuvant molecule for TMZ therapy.
** ** *
*
*
50
60
70
80
90
100
50µM 100µM 250µM
Ce
ll v
iab
ilit
y (
% f
rom
co
ntr
ol)
24 h
48 h
72 h
TMZ
Chapter IV - Results and discussion
53
After the first trial for the conditions to be used, GL261 were then incubated with
TMZ alone (250 µM) or in the presence of GUDCA (50 µM) during 24 and 72h. As
observed in Fig. IV.8, treatment with GUDCA alone caused no changes in cell
viability at both time points, as it was expected. Also anticipated was the significant
decrease induced by TMZ, a little more pronounced when time of incubation
changed from 24 (19%, p<0.01) to 72 h (31%, p<0.01). A parallel study
accomplished with the U-118 MG grade III human glioma cell line obtained a
reduction of 43% in the cell proliferation at 72 h and with 250 µM TMZ (Carmo et al.,
2011). However, and this is relevant, the incubation of TMZ in the presence of
GUDCA largely enhanced the effect of TMZ alone, further reducing the cell in 31% at
24 h and in 17% at 72h (p<0.05).
Fig. IV.8. Cell viability of GL261 cells in the absence (control) or in the presence of temozolomide
(TMZ), glycoursodeoxycholic acid (GUDCA), and TMZ+GUDCA. Glioma cells were treated with 250
μM TMZ or 50 μM GUDCA alone or in combination during 24 h and 72 h. To assess cellular viability and
functionality, we used the MTS reduction assay. Data are expressed as percentage ± SEM of the value
obtained for the control at each incubation period. **p<0.01, *p<0.05 vs. control; #p<0.05 vs. TMZ.
The chemoresistance of GL261 after 72 h of TMZ treatment with more than 60%
cells alive, may result from the subpopulation of cells that are known to be
responsible for propagation of glioblastoma growth after chemotherapy (Chen et al.,
2012). Increased sensitization by combined therapy as here observed with GUDCA
was already indicated for other compounds, such as a blocker of base excision repair
(Montaldi and Sakamoto-Hojo, 2012), inhibitors of histone deacetylases (Ryu et al.,
2012), antiangiogenic (Den et al., 2012), autophagic (Lin et al., 2012) and non-
apoptotic inducers (Overmeyer et al., 2011), inhibitors of Mrp1 activity (Peignan et
al., 2011), among many other proposals. We may then assume that dual (or even
triple) targeting directed delivery systems are promising strategies against glioma.
Given the beneficial effects evidenced by the combined treatment TMZ+GUDCA,
we thought to next assess the effect of these treatments on the cell cycle and to
** # * **
# **
0
20
40
60
80
100
120
C 50µM GUDCA 250µM TMZ 250µM TMZ +50µM GUDCA
Ce
ll v
iab
ilit
y (
%)
24h 72h
Chapter IV – Results and discussion
54
determine whether the reduced cell viability was related to an increase in apoptosis.
The cell cycle analysis was performed by flow cytometry and in this technique the
cells to be analyzed must be very well dissociated, avoiding the formation of cell
aggregates. However, our cells formed these cellular bulks very easily, hindering the
readings and leading to the absence of results, which happened very frequently,
particularly concerning the untreated (control) cells. Therefore, in the absence of
some control values, it was very difficult to ascertain the role of TMZ in the cell cycle
of GL261 cells, which was never described. However, this effect was already studied
by other and it was described that different cell lines possess distinct TMZ effects on
cell cycle (Zhang et al., 2010), depending on their resistance this drug. In fact, while
Hirose et al indicated that TMZ led to cell cycle arrest in G2, whit an accumulation of
cells at this phase, Carmo et al reported that TMZ did not induced cell cycle arrest
nor in G1 or G2 (Carmo et al., 2011).
Due to the above described method limitations, and in the absence of trusting
control values, we decide to present our results in a way that we can ascertain about
the cell cycle effect of TMZ+GUDCA co-incubation (Table IV.1.). As in cell viability,
the more significant results were obtained at the 72h incubation period. At this
timepoint, we have observed that TMZ plus GUDCA has induced cell cycle arrest at
the G2 phase, since there is an accumulation of cells at this check point (2.7-fold
increase vs. TMZ alone, p<0.01). Consequently, the number of cells at the S phase
have further decreased has compared to TMZ (0.6-fold, p<0.05). Interestingly, similar
results were obtained by Yuan et al, that found that the combination of resveratrol
and TMZ significantly resulted in G2/M cell cycle arrest in a model of GBM (Yuan et
al., 2012). Since GUDCA is also an antioxidant molecule, we may speculate that it
may share some of the already mechanisms by which resveratrol increase TMZ
efficacy.
Nonetheless, cell cycle of GL261 cells after treatments with either GUDCA or
TMZ alone or TMZ in the presence of GUDCA were characterized by a low apoptotic
fraction (data not shown), which prove that the reduction of cell viability is not related
with apoptosis. Due to the described limitations of the used cell cycle analysis
method, further assays should be performed using other procedures to analyse the
cell cycle, such as the expression of some cell cycle related proteins.
Chapter IV - Results and discussion
55
Table IV.1. Cell cycle analysis of GL261 cells incubated in the presence of temozolomide
(TMZ), glycoursodeoxycholic acid (GUDCA) and TMZ+GUDCA, during 24 and 72.
Incubation conditions
Cell cycle phase (fold change from TMZ)
G0/G1 S G2/M
24h 72h
TMZ GUDCA TMZ+GUDCA TMZ GUDCA TMZ+GUDCA
1.00±0.37 0.97±0.02 0.79±0.01
1.00±0.18 1.46±0.10 1.03±0.08
1.00±0.01 0.89±0.01** 0.98±0.001
1.00±0.13
0.74±0.05* 0.63±0.07*
1.00±0.01 0.61±0.05**
1.09±0.02
1.00±0.49 0.66±0.15
2.66±0.44*
Glioma cells were treated with TMZ (250 μM) alone or in the presence of GUDCA (50 μM) during 24
and 72 h. Results are expressed as mean fold change from TMZ±SD). **p<0.01 and *p<0.05 vs. TMZ.
Overall, we can postulate that GUDCA can be useful as an adjuvant molecule for
TMZ, particularly when used in therapeutic schemes with longer administration
periods.
3.3. Effect of TMZ, MK-571 and TMZ+MK571 on glioma cells viability and cell
cycle
Mrp1 transporters have been associated to drug efflux and resistance to
chemotherapy in high-grade gliomas, showing an average expression of 51.3% in
glioma specimens. Moreover, as no changes were detected between primary or
recurrent gliomas, it is suggest that chemoresistance is mostly intrinsic (Calatozzolo
et al., 2005), therefore, strategies to decrease the expression of the MRP gene have
been enlightened (Matsumoto et al., 2004). In line with this, the inhibitor
indomethacin has shown to significantly increase the cytotoxic effect of etoposide,
and even more that of vincristine (Benyahia et al., 2004). MK-571 is a specific Mrp1
inhibitor, which may improve TMZ treatment, as it did for vincristine and etoposide
(Peignan et al., 2011). However, these authors did not observe a beneficial effect by
the combination of MK571 and TMZ when working with T98G and G44 GBM cell
lines and suggest that TMZ may not be a substrate for Mrp1. However they only
used TMZ at 100 µM and MK-571 (20 µM) for 24 h. As we showed in Fig. IV.7, this
concentration of TMZ only slightly decreased the cell viability of our GL261 cell line.
Therefore, we decided to test the combined effects of TMZ+MK-571 in our model.
GL261 cells were treated (or untreated) with 250 µM TMZ alone or TMZ in the
presence of MK-571 (25 µM) during 24 and 72 h, as pre-established. Once again,
like GUDCA, MK-571 appears to be innocuous for the cells (Fig. IV.9), but caused a
Chapter IV – Results and discussion
56
significant reduction in cell viability when associated to TMZ. Thus, the effect caused
by TMZ treatment in the inhibition of MTS reduction by GL261 cells, already
previously noticed, was here potentiated in a dose- and time-dependent manner in
the presence of MK-571 at 24 h (~8% more vs. TMZ alone, p<0.05) increasing at 72h
(~14% more, vs. TMZ alone, p<0.05).
Fig. IV.9 Cell viability of GL261 cells in the absence (control) or in the presence of temozolomide
(TMZ), Mrp1 inhibitor (MK-571) and TMZ+MK-571. Glioma cells were treated with 250 μM TMZ or 25
μM MK-571 alone, or in combination during 24 and 72 h. To assess cellular viability and functionality,
we used the MTS reduction assay. Data are expressed as percentage ± SEM of the value obtained for
the control at each incubation period. . **p<0.015 and *p<0.05 vs. control; #p<0.05 vs. TMZ alone.
Once more, after cell viability assay, we have studied the effect of MK-571 and
TMZ co-incubation in the cell cycle, using the same approach used for GUDCA
studies, regarding the presentation of the results (Table IV.2). Similarly to GUDCA,
higher and more significant variations were obtained at the 72h incubation period.
We have observed that TMZ and GUDCA co-incubation provoked cell cycle arrest at
the G2/M phase, which is observed by the accumulation of cells (2.8-fold increase
vs. TMZ alone, p<0.01). Consequently, the number of cells at the S phase have
further decreased has compared to TMZ (0.6-fold, p<0.01). Once more, this additive
effect of MK-571 is not related with an increase in apoptotic cells (data not shown).
Overall, these results suggest that inhibition of Mrp1 transporter may enhance
TMZ therapy, its efficacy, particularly when used in therapeutic schemes with longer
administration periods, as postulated for GUDCA.
Gliomas comprise of significant cell heterogeneity that contains a number of
cancer stem-like cells that may contribute to the resistance to treatment. These cells
are phenotypically similar to the normal stem cells of the corresponding tissue of
origin, but they exhibit dysfunctional patterns of self-renewal and differentiation (Jin
et al., 2010) and have more growing ability during chemotherapy than that of
glioblastoma cells.
* # ** ** #
**
0
20
40
60
80
100
120
C 25µM Mk-571 250µM TMZ 250µM TMZ +25µM Mk-571
Ce
ll v
iab
ilit
y (
%)
24h 72h
Chapter IV - Results and discussion
57
Table IV.2. Cell cycle analysis of GL261 cells incubated in the presence of temozolomide (TMZ),
MK-571 and TMZ+GUDCA, during 24 and 72.
Incubation conditions
Cell cycle phase (fold change from TMZ)
G0/G1 S G2/M
24h 72h
TMZ MK-571 TMZ+MK-571 TMZ MK-571 TMZ+MK-571
1.00±0.37 0.97±0.02 0.75±0.02
1.00±0.18
1.60±0.07** 1.04±0.09
1.00±0.01 0.85±0.02**
1.00±0.09
1.00±0.13 0.63±0.03** 0.62±0.11**
1.00±0.01 0.79±0.01**
1.14±0.24
1.00±0.49 0.70±0.27
2.81±0.36**
Glioma cells were treated with TMZ (250 μM) alone or in the presence of MK-571 (25 μM) during 24 and
72 h. Results are expressed asmean fold change from TMZ±SD). **p<0.01 and *p<0.05 vs. TMZ.
As it was already demonstrated that these glioblastoma stem-like cells have a
higher Mrp1 expression than Mrp1 (Jin et al., 2010), we can speculate that the
inhibition of this ABC transporter may be an important tool to decrease this
chemoresistance phenomenon, which will open important avenues regarding glioma
therapy.
However, it should be interesting to further investigate the role of Mrp1 in the
GL261 cell line. This way, further assays should include the study of the Mrp1 activity
in glioma cells and the respective effect of TMZ and TMZ plus MK-571, to ascertain
about the level of Mrp1 inhibition activity achieved by MK-571. Further studies on
Mrp1 modulation can also include Mrp1 silencing in glioma cells.
4. Effect of GUDCA and MK-571 in tumor cell migration
Briefly, the results obtained in the previous assays showed that either GUDCA or
the Mrp1 inhibitor, MK-571, in addition to TMZ, enhanced the efficacy of TMZ
treatment alone. Taking this in account, we considered that it would be important to
elucidate which mechanism is underlying the potentiation of TMZ therapy by these
compounds. For that, we proceed to analyze the expression of CXCR4, known to
promote motility and proliferation of glioma cells (Carmo et al., 2010), in an attempt
to clarify if it modulates cell migration. For this, GL261 cells with 3, 5 or 7 DIV were
treated with either GUDCA or MK-571, during 24 and 72 h, using the same
concentrations of previous assays (Fig. IV.10).
Chapter IV – Results and discussion
58
0
1
2
3
4
C GU Mk 25
CX
CR
4 e
xp
ress
ion
(
fold
ch
an
ge
fro
m c
on
tro
l) 24h 72h
CXCR4
β-actin
47 kDa
42 kDa
C GUDCA MK-571
CXCR4
β-actin
47 kDa
42 kDa
24 h
72 h
C GUDCA MK-571
C GUDCA MK-571
GL261 3DIV
0
1
2
3
4
C GU Mk 25
CX
CR
4 e
xp
ress
ion
(f
old
ch
an
ge
fro
m c
on
tro
l)
CXCR4
β-actin
47 kDa
42 kDa
C GUDCA MK-571
CXCR4
β-actin
47 kDa
42 kDa
C GUDCA MK-571
24 h
72 h
C GUDCA MK-571
GL261 5DIV
0
1
2
3
4
C GU Mk 25
CX
CR
4 e
xp
ress
ion
(f
old
ch
an
ge
fro
m c
on
tro
l)
CXCR4
β-actin
47 kDa
42 kDa
C GUDCA MK-571
CXCR4
β-actin
47 kDa
42 kDa
C GUDCA MK-571
24 h
72 h
C GUDCA MK-571
GL261 7DIV
A)
B)
C)
Fig. IV.10. CXCR4 expression in glioma cells at 3, 5 and 7 days in vitro in the absence
(control) or in the presence of either glycoursodeoxycholic acid (GUDCA) ot the Mrp1 inhibitor
MK-571. Glioma cells were not-treated or treated with 50 μM GUDCA or 25 μM MK-571 during 24 and
72 h. Total cell lysates were analyzed by Western blotting with an antibody specific for CXCR4. Results
of analysis of 3, 5 and 7 DIV (A, B and C, respectively) are shown by representative results from one
experiment.and graph bars representing the intensity of the bands quantified by scanning densitometry,
standardized with respect to β-actin protein and expressed as mean ± SEM fold change from respective
control (accepted as 1).
Chapter IV - Results and discussion
59
We have observed that CXCR4 was constitutively expressed in GL261 cells, as
demonstrated in other cell glioma lines (Carmo et al., 2010). Both compounds,
GUDCA and MK-571 seem to induce the expression of CXCR4, mainly after 72 h
incubation. While GUDCA evidence a preferential induction on glioma cells at 3 DIV,
decreasing thereafter (from 2.1-fold to 0.8-fold vs. control), MK-571 suggests more
efficacy on GL261 at 7 DIV. Although only two experiments were performed, both
GUDCA and MK-571 revealed to have no effect if used for 24 h, but indeed induced
(despite not significantly) an increase in the expression of CXCR4. Therefore, none
of the compounds tested showed to be CXCR4 antagonists, what is usually looked
for glioma therapy (Terasaki et al., 2011; Fareh et al., 2012; Yu et al., 2012).
However, due to other beneficial effects, they can be given for periods that do not
surpass the 24 h. Anyway, since only 2 assays were performed we still should
confirm the results acquired and determine whether the chemokine to this receptor,
the CXCL12 (also stroma-derived factor 1. SDF-1) is actually released. This feature
is relevant once it was demonstrated an alternative receptor, the CXCR7 with a 10
times higher affinity for SDF-1 (Balabanian et al., 2005) and also controls cell
proliferation and migration (Odemis et al., 2012).
Moreover, there are studies showing that CXCL12 alone cannot induce glioma
formation, and that CXCR4 inhibition does not attenuate gliomagenesis in a mouse
model of Neurofibromatosis-1 (NF1)-associated optic pathway glioma (OPG) (Sun et
al., 2010). Re-evaluation of the roles of GUDCA and MK-571 on CXCR7 and CXCR4
will be then important as targets to be modulated by therapy to glioma. Additionally it
will be interesting to evaluate the effects of both compounds on MMPs activity once
we saw that they were increasingly expressed at 3 DIV by GL261 cells (Fig. IV.2)
and its action is mediated through the SDF-1/CXCR4 axis (Bhoopathi et al., 2011).
Chapter IV – Results and discussion
60
61
CHAPTER IV – Concluding remarks
62
Chapter VI - Concluding remarks
63
The study of tumorigenesis and the evaluation of new therapies for glioma
require accurate brain tumor models. Cultures of malignant cells represent an
excellent and permanent material for studying the biology of these tumors as, for
example, specific antigens characterization, bioactive factors produced,
determination of cellular proliferation, as well as heterogeneity of genotypic and
phenotypic characteristics (Machado et al., 2005). Our GL261 glioma cell line
characterization demonstrated that the cells express both undifferentiated (as
Vimentin, Nestin, Sox2, as well βIII-Tubulin) and differentiated proteins (as GFAP,
GLAST and MAP2), which levels change during the time in culture (Fig. V.1). This is
in agreement with the cellular heterogeneity found in other glioma cell lines
containing cells at different stages of differentiation (Shiras et al., 2003; Gangemi et
al., 2009; Zhang et al., 2011) and to what was observed in human glioma (Bonavia et
al., 2011). Therefore, this cell line has shown suitable characteristics for cellular and
molecular studies, as well as for new trearment strategies for glioma, reason why it
was used in the present study.
In this study, we have evaluated the GL261 cells characteristics throughout time
in culture, which is unusual to find in the literature, but it might be fundamental to
better understand in which way cell markers and certain essential features progress
along tumor cell differentiation. In fact, we hypothesize that a good cell
characterization will allow the correlation of the grade of maturation of the cells with
the grade of malignancy. This information will be very useful for a more directed
therapeutic targeting, especially if we want to use a cell model that can mimic the
stages with highest resistance.
Regarding the evaluation of tumor-related factors either in glioma cells or in NS
and respective differentiating astrocytes, though not as evident as it was expected,
the results suggest that the initial stage, the proliferating NS, is the phenotype that
presents more similarities with tumor cells concerning the majority of the evaluated
tumor factors (Table.V.1). Thus, this may indicate that gliomagenesis could be
related with malignant transformation of NSC, as suggested by many authors (Shiras
et al., 2003; Gangemi et al., 2009), which is not surprising due to the similarities
between both populations. However, based on some contradictory results along the
maturation process of glioma cells, future research on this tumorigenic process is
needed in order to confirm such hypothesis. Thus, our initial thoughts were to induce
a glioma phenotype in both NSC and differentiating astrocytes to look which
produced phenotype would be closer and acquire the same tumorigenic properties of
glioma cells. This cell transformation could be performed by silencing both for the
RNA of the lipid phosphatase PTEN and the tumor suppressor factor p53 by using
Chapter VI – Concluding remarks
64
the respective siRNA, as it is described that this double inactivation cooperates to
induce high-grade malignant gliomas (Zheng et al., 2008).
Fig. V.1. Summary of GL261 cell line characterization. Schematic representation of the cellular
markers expression along time in culture (3, 5 and 7 days in vitro – DIV). Throughout time, there was a
variable expression of these markers. Sox2 and βIII-tubulin expression decreased from 3 to 7 DIV, while
vimentin increased till 5 DIV and then remained constant. GFAP (glial fibrillary acidic protein) suffered a
reduction from 5 to 7 DIV, while GLAST (glutamate aspartate transporter) and MAP2 (microtubule-
associated protein 2) revealed a peak expression level at 5 DIV.
Treatment of glioma cells with TMZ in the presence of GUDCA or MK-571 greatly
enhanced the effect of TMZ alone, causing a further loss of cell viability, specially at
72 h. In addition, at the same conditions, it was observed an accumulation of cells at
G2/M phase, corresponding to a cell cycle arrest at this checkpoint. Moreover, both
co-incubation schemes showed low apoptotic levels, which indicates that the
decrease of cell viability is not correlated with this type of cell death. Overall, our
results suggest that GUDCA and MK-571 can act as adjuvants of TMZ therapy,
particularly when used in therapeutic schemes with longer administration periods.
Chapter VI - Concluding remarks
65
Intriguingly, either GUDCA or MK-571 seems to improve the migratory ability of
GL261, by the induction of increase of CXCR4 levels, although with distinct patterns.
Table. V.1. Characterization of common features between GL261 glioma cells and neurospheres
induced to differentiate into astrocytes during 3 and 7 days in vitro.
The expression of the tumor-related factors is represented from the lowest (+) to the highest expression
(+++). The green, red and blue squares represent, for each evaluated factor, the phenotype more
similar to neurospheres (NS) or to 3 and 7 DIV differentiating astrocytes. The analysis of tumor-related
factors showed that during GL261 maturation, there is a decrease on the expression of the vascular
endothelial growth factor (VEGF) as well as on the activity of the matrix metalloproteinases MMP-9 and
MMP-2, which is associated with an increase on S100B release. Also, the Mrp1 presents a peak of
expression at 5 DIV. Overall, but not as evident as we expected. NS is the phenotype that present the
highest similarities with GL261 cells. LC3, light chain 3, LC3II/LC3I ratio; Mrp1, multidrug resistance-
associated protein 1; MMPs, matrix metalloproteinases; VEGF, vascular endothelial growth factor.
Due to these contradictory findings, the therapeutic potential of GUDCA and
Mrp1 modulation should be further investigated by using in vivo studies. Thus, in a
near future we propose to use these molecules in association with TMZ in an in vivo
model of glioma, where tumors will be generated by intracerebral implantation of the
GL261 glioma cells in rats (Doblas et al., 2010). We anticipate that these therapeutic
schemes will inhibit tumor cell growth and prevent vascular alterations in early stages
of glioma progression.
Interestingly, the factors with highest impact face to non-glioma cells were
S100B, VEGF and Mrp1, in agreement with their fundamental role in glioma
development. Taking this in account, these factors may be potential therapeutic
targets and, subsequently, it would be interesting to further evaluate the synergistic
effect of TMZ and GUDCA or MK-571 on these tumor related features.
Chapter VI – Concluding remarks
66
The overall goal is to translate these results into the clinical practice, which will
reduce the resistance to the commonly used chemotherapeutic drugs, increasing the
survival of brain tumor patients.
67
Chapter VI - References
68
Chapter VI - References
69
ABTA (2010). Brain Tumor Primer - A Comprehensive IntroductIon to Brain tumors. Akare, S., S. Jean-Louis, et al. (2006). "Ursodeoxycholic acid modulates histone
acetylation and induces differentiation and senescence." Int J Cancer 119(12): 2958-2969.
Allen, C., M. Opyrchal, et al. (2012). "Oncolytic measles virus strains have significant antitumor activity against glioma stem cells." Gene Ther. 1476-5462
Annabi, B., S. Rojas-Sutterlin, et al. (2008). "Tumor environment dictates medulloblastoma cancer stem cell expression and invasive phenotype." Mol Cancer Res 6(6): 907-916.
Argyriou, A. A., A. Antonacopoulou, et al. (2009). "Treatment options for malignant gliomas, emphasizing towards new molecularly targeted therapies." Crit Rev Oncol Hematol 69(3): 199-210.
Atkinson, J. M., R. J. Gilbertson, et al. (2009). "Brain cancer stem cells as targets of novel therapies." CNS Cancer: 1057-1075.
Baber, Z. and N. Haghighat (2010). "Glutamine synthetase gene expression and glutamate transporters in C6-glioma cells." Metab Brain Dis 25(4): 413-418.
Bachoo, R. M., E. A. Maher, et al. (2002). "Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis." Cancer Cell 1(3): 269-277.
Balabanian, K., B. Lagane, et al. (2005). "The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes." J Biol Chem 280(42): 35760-35766.
Bao, S., Q. Wu, et al. (2006). "Glioma stem cells promote radioresistance by preferential activation of the DNA damage response." Nature 444(7120): 756-760.
Begley, D. J. (2004). "ABC transporters and the blood-brain barrier." Curr Pharm Des 10(12): 1295-1312.
Beier, D., P. Hau, et al. (2007). "CD133+ and CD133- glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles." Cancer research 67(9): 4010.
Benyahia, B., S. Huguet, et al. (2004). "Multidrug resistance-associated protein MRP1 expression in human gliomas: chemosensitization to vincristine and etoposide by indomethacin in human glioma cell lines overexpressing MRP1." J Neurooncol 66(1-2): 65-70.
Bhoopathi, P., C. Chetty, et al. (2011). "MMP-2 mediates mesenchymal stem cell tropism towards medulloblastoma tumors." Gene Ther 18(7): 692-701.
Bi, C. L., J. S. Fang, et al. (2007). "[Chemoresistance of CD133(+) tumor stem cells from human brain glioma]." Zhong Nan Da Xue Xue Bao Yi Xue Ban 32(4): 568-573.
Bianchi, R., E. Kastrisianaki, et al. (2011). "S100B protein stimulates microglia migration via RAGE-dependent up-regulation of chemokine expression and release." J Biol Chem 286(9): 7214-7226.
Blumcke, I., A. J. Becker, et al. (2001). "Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells." J Neuropathol Exp Neurol 60(10): 984-993.
Bonavia, R., M. M. Inda, et al. (2011). "Heterogeneity maintenance in glioblastoma: a social network." Cancer Res 71(12): 4055-4060.
Brito, M. A., S. Lima, et al. (2008). "Bilirubin injury to neurons: contribution of oxidative stress and rescue by glycoursodeoxycholic acid." Neurotoxicology 29(2): 259-269.
Brozzi, F., C. Arcuri, et al. (2009). "S100B protein regulates astrocyte shape and migration via interaction with Src Kinase: Implications for astrocyte development, activation, and tumor growth." J Biol Chem 284(13): 8797-8811.
Chapter VI - References
70
Calatozzolo, C., A. Canazza, et al. (2011). "Expression of the new CXCL12 receptor, CXCR7, in gliomas." Cancer Biol Ther 11(2): 242-253.
Calatozzolo, C., M. Gelati, et al. (2005). "Expression of drug resistance proteins Pgp, MRP1, MRP3, MRP5 and GST-pi in human glioma." J Neurooncol 74(2): 113-121.
Calatozzolo, C., B. Pollo, et al. (2012). "Multidrug resistance proteins expression in glioma patients with epilepsy." J Neurooncol. 110(1):129-35
Canoll, P. and J. E. Goldman (2008). "The interface between glial progenitors and gliomas." Acta Neuropathol 116(5): 465-477.
Carmo, A., H. Carvalheiro, et al. (2011). "Effect of temozolomide on the U-118 glioma cell line." Oncol Lett 2(6): 1165-1170.
Carmo, A., I. Patricio, et al. (2010). "CXCL12/CXCR4 promotes motility and proliferation of glioma cells." Cancer Biol Ther 9(1): 56-65.
Chen, J., Y. Li, et al. (2012). "A restricted cell population propagates glioblastoma growth after chemotherapy." Nature 488(7412): 522-526.
Choe, G., J. K. Park, et al. (2002). "Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype." Clin Cancer Res 8(9): 2894-2901.
Cole, S. P., G. Bhardwaj, et al. (1992). "Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line." Science 258(5088): 1650-1654.
Dahlstrand, J., V. P. Collins, et al. (1992). "Expression of the class VI intermediate filament nestin in human central nervous system tumors." Cancer Res 52(19): 5334-5341.
Dean, M., T. Fojo, et al. (2005). "Tumour stem cells and drug resistance." Nat Rev Cancer 5(4): 275-284.
Deleyrolle, L. P., M. R. Rohaus, et al. (2012). "Identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (CFSE)." J Vis Exp(62).
Den, R. B., M. Kamrava, et al. (2012). "A phase I study of the combination of sorafenib with temozolomide and radiation therapy for the treatment of primary and recurrent high-grade gliomas." Int J Radiat Oncol Biol Phys. 3016(12).
Dirks, P. B. (2008). "Brain tumour stem cells: the undercurrents of human brain cancer and their relationship to neural stem cells." Philos Trans R Soc Lond B Biol Sci 363(1489): 139-152.
Doblas, S., T. He, et al. (2012). "In vivo characterization of several rodent glioma models by 1H MRS." NMR Biomed 25(4): 685-694.
Doblas, S., T. He, et al. (2010). "Glioma morphology and tumor-induced vascular alterations revealed in seven rodent glioma models by in vivo magnetic resonance imaging and angiography." J Magn Reson Imaging 32(2): 267-275.
Donato, R., B. R. Cannon, et al. (2012). "Functions of S100 proteins." Curr Mol Med. Fan, Q. W., C. Cheng, et al. (2010). "Akt and autophagy cooperate to promote
survival of drug-resistant glioma." Sci Signal 3(147): ra81. Fareh, M., L. Turchi, et al. (2012). "The miR 302-367 cluster drastically affects self-
renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network." Cell Death Differ 19(2): 232-244.
Foreman, K. E., P. Rizzo, et al. (2009). "The cancer stem cell hypothesis." Stem Cells and Cancer: 1-12.
Friedman, H. S., T. Kerby, et al. (2000). "Temozolomide and treatment of malignant glioma." Clin Cancer Res 6(7): 2585-2597.
Fu, J., Z. G. Liu, et al. (2009). "Glioblastoma stem cells resistant to temozolomide-induced autophagy." Chin Med J (Engl) 122(11): 1255-1259.
Chapter VI - References
71
Furnari, F. B., T. Fenton, et al. (2007). "Malignant astrocytic glioma: genetics, biology, and paths to treatment." Genes Dev 21(21): 2683-2710.
Galli, R., E. Binda, et al. (2004). "Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma." Cancer Res 64(19): 7011-7021.
Gangemi, R. M., F. Griffero, et al. (2009). "SOX2 silencing in glioblastoma tumor-initiating cells causes stop of proliferation and loss of tumorigenicity." Stem Cells 27(1): 40-48.
Guldutuna, S., M. Leuschner, et al. (1993). "Cholic acid and ursodeoxycholic acid therapy in primary biliary cirrhosis. Changes in bile acid patterns and their correlation with liver function." Eur J Clin Pharmacol. 45(3):221-5.
Gunther, W., E. Pawlak, et al. (2003). "Temozolomide induces apoptosis and senescence in glioma cells cultured as multicellular spheroids." Br J Cancer 88(3): 463-469.
Hagemann, C., J. Anacker, et al. (2012). "A complete compilation of matrix metalloproteinase expression in human malignant gliomas." World J Clin Oncol 3(5): 67-79.
Hemmati, H. D., I. Nakano, et al. (2003). "Cancerous stem cells can arise from pediatric brain tumors." Proc Natl Acad Sci U S A 100(25): 15178-15183.
Hernandez-Garcia, D., C. D. Wood, et al. (2010). "Reactive oxygen species: A radical role in development?" Free Radic Biol Med 49(2): 130-143.
Hirose, Y., M. S. Berger, et al. (2001). "p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells." Cancer Res 61(5): 1957-1963.
Holland, E. C., J. Celestino, et al. (2000). "Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice." Nat Genet 25(1): 55-57.
Hu, L., J. Zhang, et al. (2010). "Biological characteristics of a specific brain metastatic cell line derived from human lung adenocarcinoma." Med Oncol 27(3): 708-714.
Huhn, S. L., Y. Yung, et al. (2005). "Identification of phenotypic neural stem cells in a pediatric astroblastoma." J Neurosurg 103(5 Suppl): 446-450.
Ignatova, T. N., V. G. Kukekov, et al. (2002). "Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro." Glia 39(3): 193-206.
Ihrie, R. A. and A. Alvarez-Buylla (2008). "Cells in the astroglial lineage are neural stem cells." Cell Tissue Res 331(1): 179-191.
Jain, R. K., E. di Tomaso, et al. (2007). "Angiogenesis in brain tumours." Nat Rev Neurosci 8(8): 610-622.
Jan, H. J., C. C. Lee, et al. (2010). "Osteopontin regulates human glioma cell invasiveness and tumor growth in mice." Neuro Oncol 12(1): 58-70.
Jansen, M., P. C. de Witt Hamer, et al. (2004). "Current perspectives on antiangiogenesis strategies in the treatment of malignant gliomas." Brain Res Brain Res Rev 45(3): 143-163.
Jansen, M., S. Yip, et al. (2010). "Molecular pathology in adult gliomas: diagnostic, prognostic, and predictive markers." Lancet Neurol 9(7): 717-726.
Jensen, R. L. (2009). "Brain tumor hypoxia: tumorigenesis, angiogenesis, imaging, pseudoprogression, and as a therapeutic target." J Neurooncol 92(3): 317-335.
Jiang, W., Q. Jia, et al. (2011). "S100B promotes the proliferation, migration and invasion of specific brain metastatic lung adenocarcinoma cell line." Cell Biochem Funct 29(7): 582-588.
Jimenez, A. L., A. H. Chou, et al. (2003). "Wnt-1 has multiple effects on the expression of glutamate transporters." Neurochem Int 42(4): 345-351.
Chapter VI - References
72
Jin, F., L. Zhao, et al. (2010). "Influence of Etoposide on anti-apoptotic and multidrug resistance-associated protein genes in CD133 positive U251 glioblastoma stem-like cells." Brain Res 1336: 103-111.
Jin, F., L. Zhao, et al. (2008). "Comparison between cells and cancer stem-like cells isolated from glioblastoma and astrocytoma on expression of anti-apoptotic and multidrug resistance-associated protein genes." Neuroscience 154(2): 541-550.
Joo, K. M., J. Jin, et al. (2012). "Trans-differentiation of neural stem cells: a therapeutic mechanism against the radiation induced brain damage." PLoS One 7(2): e25936.
Joo, S. S., T. J. Won, et al. (2004). "Potential role of ursodeoxycholic acid in suppression of nuclear factor kappa B in microglial cell line (BV-2)." Arch Pharm Res 27(9): 954-960.
Jouanneau, E. (2008). "Angiogenesis and gliomas: current issues and development of surrogate markers." Neurosurgery 62(1): 31-50; discussion 50-32.
Jung, C. S., C. Foerch, et al. (2007). "Serum GFAP is a diagnostic marker for glioblastoma multiforme." Brain 130(Pt 12): 3336-3341.
Kanzawa, T., I. M. Germano, et al. (2004). "Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells." Cell Death Differ 11(4): 448-457.
Kargiotis, O., J. S. Rao, et al. (2006). "Mechanisms of angiogenesis in gliomas." J Neurooncol 78(3): 281-293.
Katsetos, C. D., P. Draber, et al. (2011). "Targeting betaIII-tubulin in glioblastoma multiforme: from cell biology and histopathology to cancer therapeutics." Anticancer Agents Med Chem 11(8): 719-728.
Katsetos, C. D., E. Draberova, et al. (2009). "Tubulin targets in the pathobiology and therapy of glioblastoma multiforme. II. gamma-Tubulin." J Cell Physiol 221(3): 514-520.
Katsetos, C. D., E. Draberova, et al. (2007). "Class III beta-tubulin and gamma-tubulin are co-expressed and form complexes in human glioblastoma cells." Neurochem Res 32(8): 1387-1398.
Katsetos, C. D., M. M. Herman, et al. (2003). "Class III beta-tubulin in human development and cancer." Cell Motil Cytoskeleton 55(2): 77-96.
Katsetos, C. D., G. Karkavelas, et al. (1998). "Class III beta-tubulin isotype (beta III) in the adrenal medulla: I. Localization in the developing human adrenal medulla." Anat Rec 250(3): 335-343.
Kaur, B., F. W. Khwaja, et al. (2005). "Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and angiogenesis." Neuro Oncol 7(2): 134-153.
Kaus, A., D. Widera, et al. (2010). "Neural stem cells adopt tumorigenic properties by constitutively activated NF-kappaB and subsequent VEGF up-regulation." Stem Cells Dev 19(7): 999-1015.
Lapenna, D., G. Ciofani, et al. (2002). "Antioxidant properties of ursodeoxycholic acid." Biochem Pharmacol 64(11): 1661-1667.
Lazaridis, K. N., G. J. Gores, et al. (2001). "Ursodeoxycholic acid 'mechanisms of action and clinical use in hepatobiliary disorders'." J Hepatol 35(1): 134-146.
Lebedeva, I. V., P. Pande, et al. (2011). "Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters." PLoS One 6(7): e22429.
Leclerc, E., G. Fritz, et al. (2007). "S100B and S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains." J Biol Chem 282(43): 31317-31331.
Lee, J., H. Yu, et al. (2011). "Differential dependency of human cancer cells on vascular endothelial growth factor-mediated autocrine growth and survival." Cancer Lett 309(2): 145-150.
Chapter VI - References
73
Lee, S. W., H. Y. Cho, et al. (2012). "CD40 stimulation induces vincristine resistance via AKT activation and MRP1 expression in a human multiple myeloma cell line." Immunol Lett 144(1-2): 41-48.
Leslie, E. M., R. G. Deeley, et al. (2001). "Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters." Toxicology 167(1): 3-23.
Lin, C.-J., C.-C. Lee, et al. (2012). "Resveratrol enhances the therapeutic effect of temozolomide against malignant glioma in vitro and in vivo by inhibiting autophagy." Free Radic Biol Med 52(2): 377-391.
Lin, C. J., C. C. Lee, et al. (2012). "Inhibition of mitochondria- and endoplasmic reticulum stress-mediated autophagy augments temozolomide-induced apoptosis in glioma cells." PLoS One 7(6): e38706.
Lin, C. J., C. C. Lee, et al. (2012). "Resveratrol enhances the therapeutic effect of temozolomide against malignant glioma in vitro and in vivo by inhibiting autophagy." Free Radic Biol Med 52(2): 377-391.
Liu, Y., Y. Zhou, et al. (2011). "Expression of VEGF and MMP-9 and MRI imaging changes in cerebral glioma." Oncol Lett 2(6): 1171-1175.
Llaguno, S. A., J. Chen, et al. (2008). Neural and cancer stem cells in tumor suppressor mouse models of malignant astrocytoma. Cold Spring Harbor
Symposia on Quantitative Biology. 73: 421-426 Louis, D. N., H. Ohgaki, et al. (2007). "The 2007 WHO classification of tumours of the
central nervous system." Acta Neuropathol 114(2): 97-109. Lu, D. Y., Y. M. Leung, et al. (2010). "Glial cell line-derived neurotrophic factor
induces cell migration and matrix metalloproteinase-13 expression in glioma cells." Biochem Pharmacol 80(8): 1201-1209.
Ma, X., K. Yoshimoto, et al. (2012). "Associations between microRNA expression and mesenchymal marker gene expression in glioblastoma." Neuro Oncol 14(9): 1153-1162.
Machado, C. M., A. Schenka, et al. (2005). "Morphological characterization of a human glioma cell l ine." Cancer Cell Int 5(1): 13.
Macintosh, R. L., P. Timpson, et al. (2012). "Inhibition of autophagy impairs tumor cell invasion in an organotypic model." Cell Cycle 11(10): 2022-2029.
Mason, W. P. and J. G. Cairncross (2005). "Drug Insight: temozolomide as a treatment for malignant glioma--impact of a recent trial." Nat Clin Pract Neurol 1(2): 88-95.
Matsumoto, Y., K. Miyake, et al. (2004). "Reduction of expression of the multidrug resistance protein (MRP)1 in glioma cells by antisense phosphorothioate oligonucleotides." J Med Invest 51(3-4): 194-201.
Mennel, H. D. and B. Lell (2005). "Ganglioside (GD2) expression and intermediary filaments in astrocytic tumors." Clin Neuropathol 24(1): 13-18.
Merkle, F. T. and A. Alvarez-Buylla (2006). "Neural stem cells in mammalian development." Curr Opin Cell Biol 18(6): 704-709.
Miller, F. D. and A. Gauthier-Fisher (2009). "Home at last: neural stem cell niches defined." Cell Stem Cell 4(6): 507-510.
Montaldi, A. P. and E. T. Sakamoto-Hojo (2012). "Methoxyamine sensitizes the resistant glioblastoma T98G cell line to the alkylating agent temozolomide." Clin Exp Med.
Nicolis, S. K. (2007). "Cancer stem cells and "stemness" genes in neuro-oncology." Neurobiol Dis 25(2): 217-229.
Odemis, V., J. Lipfert, et al. (2012). "The presumed atypical chemokine receptor CXCR7 signals through G(i/o) proteins in primary rodent astrocytes and human glioma cells." Glia 60(3): 372-381.
Ogasawara, M. A. and H. Zhang (2009). "Redox regulation and its emerging roles in stem cells and stem-like cancer cells." Antioxid Redox Signal 11(5): 1107-1122.
Chapter VI - References
74
Ohgaki, H. (2009). "Epidemiology of brain tumors." Methods Mol Biol 472: 323-342. Ohgaki, H. and P. Kleihues (2005). "Epidemiology and etiology of gliomas." Acta
Neuropathol 109(1): 93-108. Omay, S. B. and M. A. Vogelbaum (2009). "Current concepts and newer
developments in the treatment of malignant gliomas." Indian J Cancer 46(2): 88-95.
Oppel, F., N. Muller, et al. (2011). "SOX2-RNAi attenuates S-phase entry and induces RhoA-dependent switch to protease-independent amoeboid migration in human glioma cells." Mol Cancer 10: 137.
Overmeyer, J. H., A. M. Young, et al. (2011). "A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells." Mol Cancer 10: 69.
Palos, T. P., S. Zheng, et al. (1999). "Wnt signaling induces GLT-1 expression in rat C6 glioma cells." J Neurochem 73(3): 1012-1023.
Palumbo, S., L. Pirtoli, et al. (2012). "Different involvement of autophagy in human malignant glioma cell lines undergoing irradiation and temozolomide combined treatments." J Cell Biochem 113(7): 2308-2318.
Pang, X., J. Min, et al. (2012). "S100B protein as a possible participant in the brain metastasis of NSCLC." Med Oncol.12-169
Parney, I. F. and S. M. Chang (2003). "Current chemotherapy for glioblastoma." Cancer J 9(3): 149-156.
Peignan, L., W. Garrido, et al. (2011). "Combined use of anticancer drugs and an inhibitor of multiple drug resistance-associated protein-1 increases sensitivity and decreases survival of glioblastoma multiforme cells in vitro." Neurochem Res 36(8): 1397-1406.
Perez, M. J., R. I. Macias, et al. (2006). "Maternal cholestasis induces placental oxidative stress and apoptosis. Protective effect of ursodeoxycholic acid." Placenta 27(1): 34-41.
Ponnala, S., C. Chetty, et al. (2011). "MMP-9 silencing regulates hTERT expression via beta1 integrin-mediated FAK signaling and induces senescence in glioma xenograft cells." Cell Signal 23(12): 2065-2075.
Rao, J. S. (2003). "Molecular mechanisms of glioma invasiveness: the role of proteases." Nat Rev Cancer 3(7): 489-501.
Reddy, E. M., S. T. Chettiar, et al. (2011). "Dlxin-1, a member of MAGE family, inhibits cell proliferation, invasion and tumorigenicity of glioma stem cells." Cancer Gene Ther 18(3): 206-218.
Rodrigues, C. and C. J. Steer (2001). "The therapeutic effects of ursodeoxycholic acid as an anti-apoptotic agent." 10(7):1243-53.
Rodrigues, C. M., C. L. Stieers, et al. (2000). "Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition." J Neurochem 75(6): 2368-2379.
Rothermundt, M., M. Peters, et al. (2003). "S100B in brain damage and neurodegeneration." Microsc Res Tech 60(6): 614-632.
Rudolph, G., P. Kloeters-Plachky, et al. (2002). "Intestinal absorption and biliary secretion of ursodeoxycholic acid and its taurine conjugate." Eur J Clin Invest 32(8): 575-580.
Rutka, J. T., M. Murakami, et al. (1997). "Role of glial filaments in cells and tumors of glial origin: a review." J Neurosurg 87(3): 420-430.
Ryu, C. H., W. S. Yoon, et al. (2012). "Valproic acid downregulates the expression of MGMT and sensitizes temozolomide-resistant glioma cells." J Biomed Biotechnol 2012: 987495.
Sanai, N., A. Alvarez-Buylla, et al. (2005). "Neural stem cells and the origin of gliomas." N Engl J Med 353(8): 811-822.
Chapter VI - References
75
Schoemaker, M. H., L. Conde de la Rosa, et al. (2004). "Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways." Hepatology 39(6): 1563-1573.
Serviddio, G., J. Pereda, et al. (2004). "Ursodeoxycholic acid protects against secondary biliary cirrhosis in rats by preventing mitochondrial oxidative stress." Hepatology 39(3): 711-720.
Shah, S. A., Y. Volkov, et al. (2006). "Ursodeoxycholic acid inhibits interleukin 1 beta [corrected] and deoxycholic acid-induced activation of NF-kappaB and AP-1 in human colon cancer cells." Int J Cancer 118(3): 532-539.
Shibazaki, M., C. Maesawa, et al. (2012). "Transcriptional and post-transcriptional regulation of betaIII-tubulin protein expression in relation with cell cycle-dependent regulation of tumor cells." Int J Oncol 40(3): 695-702.
Shiras, A., A. Bhosale, et al. (2003). "A unique model system for tumor progression in GBM comprising two developed human neuro-epithelial cell lines with differential transforming potential and coexpressing neuronal and glial markers." Neoplasia 5(6): 520-532.
Silva, R., C. M. Rodrigues, et al. (2001). "Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid." 34(3):402-8.
Silveira Correa, T. C., R. R. Massaro, et al. (2010). "RECK-mediated inhibition of glioma migration and invasion." J Cell Biochem 110(1): 52-61.
Singh, S. K., I. D. Clarke, et al. (2004). "Cancer stem cells in nervous system tumors." Oncogene 23(43): 7267-7273.
Singh, S. K., I. D. Clarke, et al. (2003). "Identification of a cancer stem cell in human brain tumors." Cancer Res 63(18): 5821-5828.
Sola, S., X. Ma, et al. (2003). "Ursodeoxycholic acid modulates E2F-1 and p53 expression through a caspase-independent mechanism in transforming growth factor beta1-induced apoptosis of rat hepatocytes." 278(49):48831-8 .
Stupp, R., W. P. Mason, et al. (2005). "Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma." N Engl J Med 352(10): 987-996.
Sulman, E. P., M. Guerrero, et al. (2009). "Beyond grade: molecular pathology of malignant gliomas." Semin Radiat Oncol 19(3): 142-149.
Sun, T., S. M. Gianino, et al. (2010). "CXCL12 alone is insufficient for gliomagenesis in Nf1 mutant mice." J Neuroimmunol 224(1-2): 108-113.
Sutter, R., G. Yadirgi, et al. (2007). "Neural stem cells, tumour stem cells and brain tumours: dangerous relationships?" Biochim Biophys Acta 1776(2): 125-137.
Swarnkar, S., S. Singh, et al. (2012). "Astrocyte Activation: A Key Step in Rotenone
Induced Cytotoxicity and DNA Damage." Neurochem Res.37(10): 21 78-2189. Szatmari, T., K. Lumniczky, et al. (2006). "Detailed characterization of the mouse
glioma 261 tumor model for experimental glioblastoma therapy." Cancer Sci 97(6): 546-553.
Tamajusuku, A. S., E. S. Villodre, et al. (2010). "Characterization of ATP-induced cell death in the GL261 mouse glioma." J Cell Biochem 109(5): 983-991.
Tan, B. T., C. Y. Park, et al. (2006). "The cancer stem cell hypothesis: a work in progress." Lab Invest 86(12): 1203-1207.
Temple, S. (2001). "The development of neural stem cells." Nature 414(6859): 112-117.
Terasaki, M., Y. Sugita, et al. (2011). "CXCL12/CXCR4 signaling in malignant brain tumors: a potential pharmacological therapeutic target." Brain Tumor Pathol 28(2): 89-97.
Teres, S., V. Llado, et al. (2012). "2-Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy." Proc Natl Acad Sci U S A 109(22): 8489-8494.
Chapter VI - References
76
Thakkar, D., L. Shervington, et al. (2011). "Proteomic studies coupled with RNAi methodologies can shed further light on the downstream effects of telomerase in glioma." Cancer Invest 29(2): 113-122.
Thurston, G. and J. Kitajewski (2008). "VEGF and Delta-Notch: interacting signalling pathways in tumour angiogenesis." Br J Cancer 99(8): 1204-1209.
Van Meir, E. G., C. G. Hadjipanayis, et al. (2010). "Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma." CA Cancer J Clin 60(3): 166-193.
Vazquez, P., A. I. Arroba, et al. (2012). "Atg5 and Ambra1 differentially modulate neurogenesis in neural stem cells." Autophagy 8(2): 187-199.
Villano, J. L., T. E. Seery, et al. (2009). "Temozolomide in malignant gliomas: current use and future targets." Cancer Chemother Pharmacol 64(4): 647-655.
Vos, M. J., T. J. Postma, et al. (2004). "Serum levels of S-100B protein and neuron-specific enolase in glioma patients: a pilot study." Anticancer Res 24(4): 2511-2514.
Wali, R. K., S. Khare, et al. (2002). "Ursodeoxycholic acid and F(6)-D(3) inhibit aberrant crypt proliferation in the rat azoxymethane model of colon cancer: roles of cyclin D1 and E-cadherin." Cancer Epidemiol Biomarkers Prev 11(12): 1653-1662.
Wang, L., J. Zhang, et al. (2010). "Sumoylation of vimentin354 is associated with PIAS3 inhibition of glioma cell migration." Oncotarget 1(7): 620-627.
Westphal, M. and K. Lamszus (2011). "The neurobiology of gliomas: from cell biology to the development of therapeutic approaches." Nat Rev Neurosci 12(9): 495-508.
Wilhelmsson, U., C. Eliasson, et al. (2003). "Loss of GFAP expression in high-grade astrocytomas does not contribute to tumor development or progression." 29;22(22):3407-3411 .
Wu, A., S. Oh, et al. (2008). "Persistence of CD133+ cells in human and mouse glioma cell lines: detailed characterization of GL261 glioma cells with cancer stem cell-like properties." Stem Cells Dev 17(1): 173-184.
Xie, Z. (2009). "Brain tumor stem cells." Neurochem Res 34(12): 2055-2066. Yamanaka, R. and H. Saya (2009). "Molecularly targeted therapies for glioma." Ann
Neurol 66(6): 717-729. Yan, T., K. O. Skaftnesmo, et al. (2011). "Neuronal markers are expressed in human
gliomas and NSE knockdown sensitizes glioblastoma cells to radiotherapy and temozolomide." BMC Cancer 11: 524.
Yang, H. Y., N. Lieska, et al. (1994). "Proteins of the intermediate filament cytoskeleton as markers for astrocytes and human astrocytomas." Mol Chem Neuropathol 21(2-3): 155-176.
Ye, Z. C., J. D. Rothstein, et al. (1999). "Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters and enhanced activity of cystine-glutamate exchange." J Neurosci 19(24): 10767-10777.
Yu, K., Z. Chen, et al. (2012). "Tetramethylpyrazine-mediated suppression of C6 gliomas involves inhibition of chemokine receptor CXCR4 expression." Oncol Rep 28(3): 955-960.
Yuan, Y., X. Xue, et al. (2012). "Resveratrol enhances the antitumor effects of temozolomide in glioblastoma via ROS-dependent AMPK-TSC-mTOR signaling pathway." CNS Neurosci Ther 18(7): 536-546.
Zhang, J., M. F. Stevens, et al. (2010). "Acquired resistance to temozolomide in glioma cell lines: molecular mechanisms and potential translational applications." Oncology 78(2): 103-114.
Zhang, L., W. Liu, et al. (2011). "S100B attenuates microglia activation in gliomas: possible role of STAT3 pathway." Glia 59(3): 486-498.
Chapter VI - References
77
Zhang, S. J., F. Ye, et al. (2011). "Comparative study on the stem cell phenotypes of C6 cells under different culture conditions." Chin Med J (Engl) 124(19): 3118-3126.
Zhao, J. X., L. P. Yang, et al. (2007). "Gelatinolytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 in rat brain after implantation of 9L rat glioma cells." Eur J Neurol 14(5): 510-516.
Zhao, Y., Q. Huang, et al. (2010). "Autophagy impairment inhibits differentiation of glioma stem/progenitor cells." Brain Res 1313: 250-258.
Zheng, H., H. Ying, et al. (2008). "p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation." Nature 455(7216): 1129-1133.
Zheng, W. E., H. Chen, et al. (2012). "Overexpression of betaIII-tubulin and survivin associated with drug resistance to docetaxel-based chemotherapy in advanced gastric cancer." J BUON 17(2): 284-290.
Zhuang, W., B. Li, et al. (2011). "Induction of autophagy promotes differentiation of glioma-initiating cells and their radiosensitivity." Int J Cancer 129(11): 2720-2731.